Probing the τ Subunit and β2 Sliding

Clamp Interaction

Maialen Aizpurua Alti

School of Chemistry

Distinguished Professor Nick Dixon

2016

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS………………………………………………………………...…..iv

ABBREVIATIONS……………………………………………………………………………...v

ABSTRACT……………………………………………………………………………....……vii

1 INTRODUCTION……………………………………………………………………………1

1.1 E. COLI DNA REPLICATION………………………………………………...……….1

1.1.1 Initiation phase of E. coli DNA replication……………………………………2

1.1.2 Elongation phase of E. coli DNA replication…………………………………2

1.2 STRUCTURE AND FUNCTION OF DNA POLYMERASE III

HOLOENZYME…………………………………………………………………………4

1.2.1 The polymerase core (αεθ)…………………...……………………………….5

1.2.1.1 The α subunit……………………………………………………………………5

1.2.2 The β2 sliding clamp……………………………………………………………8

1.2.3 The clamp loader complex…………………………………………………...10

1.2.3.1 The τ and γ subunits……………………………………………………….….12

1.3 TECHNIQUES USED FOR STUDYING PROTEIN STRUCTURE AND

FUNCTION…………………………………………………………………………….14

1.3.1 DNA replication assays……………………………………………………….14

1.3.2 Mass spectrometry……………………………………………………………15

1.4 AIMS OF THIS PROJECT……………………………………………………...…....16

2 RESULTS AND DISCUSSION……………………………..……………………………18

2.1 RESULTS - τ Subunit and β2 Sliding Clamp Interaction…………………………..18

2.1.1 Cloning of mutant genes……………………………………………………...18

2.1.1.1 Double overlapping PCR for generation of full-length τ mutants…………18

2.1.1.2 Double overlapping PCR for generation of τC16 mutants………………….19

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2.1.1.3 Colony PCR on τC16 mutants in vector……………………………………20

2.1.2 Expression of τC16 mutant proteins…………………………………………..21

2.1.3 FPLC τC16 protein purification………………………………………………...22

2.1.4 Electrospray ionisation (ESI)-mass spectrometry (MS)…………………...26

2.1.5 Functional DNA replication assay…………………………………………...29

2.2 DISCUSSION………………………………………………………………………….31

2.2.1 Overproduction and purification of τC16 proteins……………………………31

2.2.2 Functional effects of τC16R561A+H562A protein…………………………..32

3 EXPERIMENTAL………………………………………………………………………….34

3.1 GENERAL MATERIALS……………………………………………………………...34

3.1.1 Oligonucleotide primers………………………………………………………34

3.1.2 Buffers………………………………………………………………………….34

3.1.3 Chemicals and reagents……………………………………………………...35

3.1.4 Bacterial strains………………………………………………………………..36

3.1.5 Plasmid vectors………………………………………………………………..36

3.1.6 Growth media………………………………………………………………….38

3.1.6.1 Lysogeny broth solid medium (agar plates)………………………………..38

3.1.6.2 Lysogeny broth liquid medium……………………………………………….38

3.2 METHODS…………………………………………………………………………..…38

3.2.1 Molecular biology methods…………………………………………………..38

3.2.1.1 Preparation of plasmid DNA by alkaline lysis………………………………38

3.2.1.2 Agarose gel electrophoresis………………………………………………….39

3.2.1.3 Polymerase chain reactions (PCR)……………………………..…………..39

a) Site-directed mutagenesis (SDM) PCR………………………………….……..39

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b) Colony PCR………………………………………………………………….……40

c) Double-overlapping PCR…………………………………………………….…..40

d) Sequencing PCR……………………………………………………………….…41

3.2.1.4 Quantification of DNA…………………………………………………………42

3.2.1.5 Restriction endonuclease digestion…………………………………………42

3.2.1.6 Purification of DNA by extraction from agarose gels………………………43

3.2.1.7 DNA ligation……………………………………………………………………43

3.2.1.8 Transformation of plasmids into competent E. coli strains………............43

3.2.1.9 Overproduction of τc16 proteins………………………………………………43

3.2.2 Protein biochemistry methods……………………………………………….44

3.2.2.1 Fast protein liquid chromatography (FPLC) purification of

proteins…………………………………………………………………………44

3.2.2.2 Dialysis of proteins…………………………………………………………….45

3.2.2.3 Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis

(PAGE)…………………………………………………………………………45

3.2.2.4 Determination of protein concentrations……………………………………45

3.2.2.5 Electrospray ionization (ESI)-mass spectrometry (MS)…………………...46

3.2.3 Bulk DNA replication assays………………………………………………....46

4 CONCLUSION……………………………………………………………………………..48

5 REFERENCES…………………………………………………………………………….50

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ACKNOWLEDGEMENTS

First of all, I would like to thank my supervisor, Prof Nick Dixon for giving me the

opportunity to be a part of such a challenging and rewarding project. Your vast scientific

knowledge and expertise is inspiring. I would also like to express my sincere thanks to

Jacob Lewis for the significant time you have invested in mentoring me. Not only did

you introduce me to the fascinating topic of DNA replication, but you also gave me

constant guidance and adequate autonomy with my work. In addition, the preliminary

work carried out by the Dixon group (particularly Dr Slobodan Jergic and Jacob Lewis)

and various collaborators has been immensely valuable. Without their efforts, my

project could not have taken off.

A big thank you to the rest of the extended Dixon, Van Oijen and Oakley group:

Dr Zhi-Qiang Xu, Dr Slobodan Jergic, Dr Fay Dawes, Dr Nan Li, Dr Andrew Robinson,

Dr Celine Kelso, Dr Flynn Hill, Dr Harshad Ghodké, Thomas, Bishnu, Nick, Varsha,

David, Kate, Amy, Han, Lisanne, Enrico, Sarah, and Richard. I had a grand time

working with you guys and I cannot express enough gratitude to you all for your

friendship, help and laughs throughout my stay. You truly have made my time in

Australia one that I will always treasure. A special thank you to my dearest friend

Varsha for believing in me through my ups and downs and always being keen for a chat

over coffee.

Also, I could not have lived this unique and exciting experience in Australia

without the opportunity given to me by my University (Cardiff University, Wales, UK).

Last but not least, I would like to thank my family for your motivation, support and

encouragement all throughout the year. You have always pushed me to pursue my

dreams even if this has meant going away from home, and I could not have gotten this

far without you.

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ABBREVIATIONS

Å Angstrom

A280 Absorbance at wavelength 280

AAA+ ATPase associated with various cellular activities

ADP adenosine diphosphate

ATP adenosine triphosphate

bp base pair(s)

DEAE diethylaminoethyl

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

dNTP deoxyribonucleoside triphosphate

ds double-stranded

DTT dithiothreitol

E. coli Escherichia coli

EDTA ethylenediaminetetraacetatic acid

ESI-MS electrospray ionization-mass spectrometry

FPLC fast protein liquid chromatography

h hour

kb kilo-bases

kDa kilo-daltons

KD equilibrium dissociation constant

LB(T)(A) Luria-Betani broth with supplement(s): (thymine) and/or (ampicillin)

LMW low molecular weight

MCS multiple cloning site

Met methionine

min minute

Mr relative molecular mass

mRNA messenger RNA

MW molecular weight

n stoichiometry

nt nucleotides

OB fold oligonucleotide/oligosaccharide-binding fold

OD600 optical density at wavelength 600

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ori origin of replication

PCR polymerase chain reaction

PMSF phenylmethylsulfonyl fluoride

Pol I DNA polymerase I

Pol III HE DNA polymerase III holoenzyme

RBS ribosome-binding site

RNA ribonucleic acid

s second

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SPR surface plasmon resonance

SSB single-stranded DNA-binding protein

ss single-stranded

TAE buffer Tris/acetate/EDTA buffer

TBE buffer Tris/borate/EDTA buffer

ts temperature-sensitive

Tris tris(hydroxymethyl)aminomethane/ Trizma

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ABSTRACT

DNA (deoxyribonucleic acid) replication is an essential process in all living

organisms where chromosomal DNA is duplicated before a cell divides into two

identical daughter cells. Multiple proteins, collectively known as the replisome, interact

through complex pathways with cellular DNA and with each other to accurately and

efficiently replicate DNA. During replication in the Escherichia coli (E. coli) replication

system, the two strands of a double-stranded DNA (dsDNA) molecule are unwound

from each other by the DnaB helicase. These strands are then used as templates for

the synthesis of two new strands by the DNA polymerase III (Pol III) holoenzyme (HE).

The Pol III HE is composed of three subassemblies – a core polymerase, a sliding

clamp and a clamp-loading complex. While two identical copies of the core polymerase

are engaged in the extension of the two new strands through nucleotide polymerisation,

the clamp-loading complex loads ring-shaped β2 sliding clamps onto DNA. β2 sliding

clamps tether the core polymerases to DNA, increasing their processivities.

The remarkably efficient replication system of E. coli is ideal for studying the

dynamic interplay among the various components of the replisome. A lot of work has

gone into deciphering the interactions between the different subunits of the Pol III HE to

gain a deeper understanding of the E. coli DNA replication machinery. The primary aim

of this project was to probe a recently discovered, novel interaction between the τ

subunit of the clamp-loading complex and the β2 sliding clamp of the E. coli DNA Pol III

HE. Mutagenesis studies were performed in which amino acid residues of τC16 (a

truncated form of τ) that were suspected of being involved in the interaction were

mutated. The τC16R561A+H562A mutant that presented the 16.37 kDa subunit

containing the double point mutations in the preferred sites was successfully purified.

The functionality of this mutant was assessed by an in vitro DNA replication assay.

However, no significant information about the probable τ–β2 interaction was provided by

this assay. Further research involving mutated residues on the complete clamp loader

is required to obtain a functional understanding of the clamp-binding site in the τ

subunit.

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1 INTRODUCTION

Cells of all organisms need to copy, or replicate, the DNA in their chromosomes

before they divide to make two daughter cells. This review will provide an insight into

the DNA replication process, specifically the role of the DNA synthesis apparatus, or

replisome in the copying of DNA in the bacterium, Escherichia coli. The structure,

function and assembly of replisomal subunits in the DNA polymerase III holoenzyme

are discussed in detail. Due to the aim of this project, the τ subunit of the clamp loader

complex and its role in replication is the main focus of this study. Furthermore, the

biochemical and analytical techniques utilised in this project to probe τ subunit

functionality are also outlined.

1.1 E. COLI DNA REPLICATION

Cellular division is an essential process for all living organisms whereby a cell

reproduces to form two genetically identical daughters. The precise transmission of

hereditary information relies on the accurate duplication of the genetic material stored in

the double helix of chromosomal DNA. This mechanism is commonly referred to as

DNA replication and it is based on the two-stranded complementary structure of the

DNA molecule. Once the parental double helix DNA is unwound by disrupting the weak

hydrogen bonds between the base pairs, the two template strands are copied

simultaneously with remarkable efficiency and accuracy. The semi-conservative fashion

of replicating the antiparallel helical structure of DNA was first modelled by Watson and

Crick in 19531. This means that each daughter cell’s double-stranded DNA (dsDNA) is

composed of a parental (template) and a complementary nascent polynucleotide

strand. To ensure the accuracy of this process, each cell contains an enzymatic

replication machinery that assembles and functions at the active sites of DNA

synthesis, known as replication forks. These large complexes, referred to as the

replisome, are involved in all coordinated protein–protein and protein–DNA interactions

necessary for the replication of DNA.

The E. coli replisome has been extensively investigated as the bacterium can be

cultivated and grown rapidly in laboratory settings. Thus, it is frequently used as a

representative for a better understanding of the replication mechanisms for other

organisms, and its genome is encoded on a single, small and circular double helical

2

DNA molecule, or chromosome (4.7 Mbp)2. The E. coli replisome copies its DNA at a

significant rate of approximately 1000 base pairs per second with an error rate as low

as one mutation per 10–9 to 10–11 bp3. The multi-protein molecular machine is

comprised of at least 13 different subunits that assemble by affinity4. The hierarchy of

strong and weak protein–protein and protein–DNA interactions is what allows the

replisome to conformationally change in order for replication to proceed in three main

steps: initiation at origin of replication (oriC), elongation of the complementary strands

at the replication fork and the termination stage at the termination (Ter) sites.

1.1.1 Initiation phase of E. coli DNA replication

Initiation of bacterial DNA replication occurs at the oriC. DnaA, a specific initiator

protein, recognizes and binds this origin5,6. Once bound, it promotes the assembly of

nucleoprotein complexes that, once activated by ATP, unwind the dsDNA to yield the

single-stranded DNA (ssDNA) bubble (“open complex”)7. DnaB6, the homohexameric

ring-shaped helicase, is then opened and loaded onto each of the single strands of the

“open complex”8,9. This loading requires the combined interactions of an ATP-activated

DnaA–oriC complex with a DnaB-bound DnaC (helicase loader) complex, which results

in the dissociation of DnaC10,11. The two helicase molecules remodel the replication

complex by moving in the 5'–3' direction on what will eventually become the lagging

strand templates of the two replication forks. This progressive translocation of the DnaB

results in the separation of the dsDNA into the leading and lagging ssDNA templates12.

Interaction of the helicase with the DnaG primase (a specialist RNA polymerase)

enables primase to synthesise RNA primers of 10–12 nucleotides (nt) complementary

to the strands on which the helicases are translocating13,14. The first primer synthesised

in each of the ssDNA at oriC becomes the leading strand primer for each replication

fork, and consecutive primers initiate DNA synthesis on the lagging strand. The

replicative polymerase, the DNA polymerase III holoenzyme (Pol III HE), is loaded at

the primer termini and its assembly to each of the replication forks allows for the start of

the elongation stage of DNA replication (Figure 1.1).

1.1.2 Elongation phase of E. coli DNA replication

Replication of the circular E. coli chromosome is bidirectional since the

translocation of the two replication forks occurs in opposite directions from oriC15. The

two complementary DNA strands have opposite polarity, which means that the leading

3

strand template runs in the 5'–3' direction while the lagging strand template runs in the

3'–5' direction. DNA polymerases synthesise DNA by elongating the 3'–OH of the

terminal nucleotide of the primer (i.e., only in the 5'–3' direction). During semi-

conservative DNA replication, both strands are replicated simultaneously by the dimeric

DNA Pol III HE located at each replication fork (Figure 1.1)16.

Figure 1.1: The E. coli chromosomal replication fork and the structure of the

replisome17. Incoming dsDNA is unwound by the progressive ATP-dependent

translocation of DnaB. The ssDNA from the lagging strand generated by helicase action

needs to be protected by SSB. Simultaneous and continuous replication of the leading

and lagging strands is enabled by the dimeric DNA Pol III HE, which is loaded at the

termini of the RNA primers synthesised by DnaG. The β2 sliding clamp subunit of the

DNA Pol III HE is assembled onto the primers by the ATPase activity of the clamp loader

complex (CLC). The CLC also provides high replication efficiency by connecting the DNA

Pol III cores (αεθ) onto the DNA. There are up to three cores in DNA Pol III HE, and they

are linked through the C-terminal domains of the τ subunits in the CLC. Due to the

opposite polarity of the complementary DNA strands, bidirectional replication is carried

out by continuous synthesis on the leading strand and discontinuous Okazaki fragment

synthesis on the lagging strand. The latter requires the reassembly of the β2-clamp and

Pol III core onto each novel RNA primer synthesised by DnaG primase in contact with

DnaB, indicating the start of a new Okazaki fragment. The Okazaki fragments are

replaced with DNA by Pol I and joined by DNA ligase.

4

The two nascent DNA strands need therefore to be synthesised by different

mechanisms, whereby the leading strand is elongated continuously while the lagging

strand is discontinuously extended in short Okazaki fragments (1000–2000 nt) that are

subsequently joined by the DNA ligase18. The lagging strand is required to form a

ssDNA loop to reorient the polymerase so it can progressively move in the same

direction as on the leading strand19. Thus, the significant amount of ssDNA in the

lagging strand generated by helicase action needs to be protected by the single-

stranded DNA-binding protein (SSB)20. This homotetrameric protein binds to ssDNA

with high affinity and in a sequence-independent fashion, enabling it to retain the

ssDNA in an appropriate conformation for the replisome to process it and to prevent its

self-annealing (e.g., hairpin formation)21. Since replicative DNA polymerases cannot

initiate a new DNA chain, each Okazaki fragment is initiated at a short RNA primer

generated by DnaG primase in contact with DnaB helicase22. A textbook model, one

that is currently being challenged, suggests that during each cycle of synthesis of a

single Okazaki fragment, a lagging-strand loop grows and then dissociates once the

lagging strand polymerase encounters the 5' end of the previous fragment. After each

Okazaki fragment synthesis, the RNA primers must be removed and replaced

simultaneously by the 5'–3' exonuclease and polymerase actions of DNA polymerase I.

This allows for the assembly of contiguous lagging-strand products by ligation of the

remaining nicks by DNA ligase23. As a result, coupled leading and the lagging strand

synthesis is progressively and simultaneously carried out by a single Pol III HE as the

replication fork invades dsDNA16. In E. coli, translocation of the replisome during this

coordinated replication process occurs at approximately 1000 base pairs per second24.

1.2 STRUCTURE AND FUNCTION OF DNA POLYMERASE III HOLOENZYME

During the last step of the initiation at oriC, DNA Pol III HE is loaded and

assembled onto each of the first leading-strand RNA primer termini. This polymerase

consists of 10 different subunits and it efficiently replicates the E. coli chromosomal

DNA with remarkable fidelity. Since one E. coli cell contains only 10–20 DNA Pol III HE

molecules, each holoenzyme must replicate the leading strand with high processivity,

remaining assembled to the primer until half of the chromosome is duplicated.

5

However, exchange or recycling of the polymerase is also required on the lagging-

strand during Okazaki fragment synthesis23.

The term holoenzyme refers to the fact that it is composed of ten different

subunits that interact and exchange by functional interactions25,26. The formula that

describes the average composition of DNA Pol III HE is (αεθ)2–(τ2γδδ’–ψχ)–(β2)2.

These subunits can be purified for in vitro studies27,28 and form stable and functionally

specific subassemblies that interconnect: the core polymerase αεθ (Pol III), the β2

homodimeric sliding clamp, and the clamp loader (CLC) or DnaX complex (τ2γδδ'–

ψχ)29. E. coli Pol III HE exhibits 5'–3' polymerase and 3'–5' exonuclease activities, and

the two core polymerases synthesise the leading and lagging strands simultaneously.

However, the Pol III core alone is neither processive (dissociates after synthesising 10–

20 nt)30 nor rapid (polymerises at a rate of ~20 nt per second)31. Once the β2

processivity factor is loaded onto the DNA by the CLC ATPase activity, the interaction

between the β2 and α subunits provides replication efficiency on all core polymerase

subassemblies32. The structure and function of the three subassemblies of the DNA Pol

III HE are described in detail in this Section.

1.2.1 The polymerase core (αεθ)

The polymerase core of the holoenzyme consists of the linear arrangement of

the α-ε-θ subunits in a 1:1:1 heterotrimer stoichiometry, where α and θ only interact with

ε, while ε interacts with both subunits33. Two (or maybe three) Pol III cores are believed

to enable simultaneous replication of both strands, with one dedicated to the synthesis

of the leading strand while the other one (or two) duplicates the lagging strand34–36.

Within a single core, the large α subunit of the Pol III core is a polymerase, the ε is a

separate 3'–5' proofreading exonuclease subunit and the small θ subunit has a role in

stabilising ε37.

1.2.1.1 The α subunit

The α subunit is a 5'–3' DNA polymerase that adds nucleotides to the 3'–OH

end of the growing strand in what is referred to as the template-directed nucleotidyl

transferase reaction. The specific properties of the E. coli α subunit are dictated by

several functional domains 38.

6

Figure 1.2: The crystal structure of a large fragment of the E. coli α subunit38. A)

The barrel representation of the α subunit. The location of the catalytically active

residues in the palm domain (Asp401, Asp403 and Asp555)39 is indicated by black spheres

and the location of the phosphate ion in the PHP domain by red spheres. B) Surface

representation of α in the presence of modelled DNA, β2 sliding clamp and proofreading

exonuclease ε.

The polymerase and histidinol-phosphatase (PHP) domain at the N-terminus

contains a trinuclear Zn2+ catalyst-dependent 3'–5' exonuclease activity that functions

as a proofreader in some organisms40. However, in E. coli Pol III, this activity has not

been detected, and instead, it has been convincingly shown to be the site of interaction

with the ε exonuclease subunit41,42 The global polymerase domain that follows the PHP

domain consists of the palm, thumb and fingers domains that characterize all DNA

polymerase enzymes43. The palm domain follows the common two metal catalytic

mechanism of the phosphoryl transfer reaction44, where two stabilising Mg2+ ions are

bound to the domain via three essential aspartic residues45. The catalytic magnesium

cation is believed to bind to the 3'–oxygen end of the growing primer while the

nucleotide metal coordinates to the triphosphate part of the incoming nucleotide. This

metal–DNA strand interaction dramatically increases the rate of dNTP incorporation via

nucleophilic attack of the 3'–OH of the primer terminus on the α-phosphate of the

incoming dNTP, with release of pyrophosphate39. The thumb domain may play a role in

positioning the nascent dsDNA and in processivity and translocation. Due to its

proximity of the palm domain, the extended fingers domain is capable of interacting with

7

incoming dNTPs. A conformational change occurs when these nucleotides are located

close to the catalytic metals, the primer strand termini and the template base to which

the domain is paired46,47.

The polymerase α subunit contains a clamp-binding motif (CBM) at the end of

the fingers domain that binds to the β2 clamp at a site near its C-terminus where many

other proteins also bind (discussed further in Section 1.2.2; see Figure 1.3)48. This

interaction has an affinity constant (KD) of 800 nM and it ensures high processivity of

the polymerase during replication. However, the Pol III core (αεθ complex) binds more

strongly to the sliding clamp (KD = 250 nM) as a result of the supplementary CBM in the

ε subunit binding to the corresponding site in the other subunit of β2. Thus, both α and ε

interact simultaneously with the β2 clamp, which maintains the αεθ–β2 replicase

complex in the polymerization mode of processive DNA synthesis42. The ε–β interaction

is weak (KD ~200 M) and hence enables other (repair or translesion) polymerases to

alternatively bind to the β2 clamp49,50. In the absence of DNA, the sliding clamp

associates strongly with the clamp loader. But once the clamp is loaded onto the

circular ssDNA, the Pol III-clamp-exonuclease trimeric complex is further stabilised (KD

< 5 nM) and out competes the clamp loader51. After replication of the template, the Pol

III loses affinity for the clamp, and the latter re-associates with the CLC.

The flexible C-terminus of the α subunit also includes an oligonucleotide/

oligosaccharide binding (OB-fold) domain that may be involved in interactions with the

ssDNA template and provides protein binding sites52,53. Ultimately, Pol III also contacts

the CLC48, which assists binding of the polymerase core onto the newly loaded β₂29.

This occurs through a strong interaction (KD ~260 pM) of the C-terminus of the α

subunit with the C-terminal domain V of the τ subunit (τC16)54.

In summation, the remarkably fast and processive enzymatic activity of the α

subunit of the E. coli polymerase relies on the sliding clamp β, the proofreading

exonuclease ε and the C-terminal domain of the clamp loader subunit τ55.

It is possible to state that the subunits in the Pol III core are coordinated and

stimulate each other’s activities within the DNA Pol III HE. The polymerase activity of α

increases 2-fold in a complex formed from α and ε subunits and that of the 3'–5'

exonuclease of ε increases 10- to 80-fold, proving that these subunits are each more

active together than separately. The stimulation of the exonuclease activity is mainly

8

due to the increased affinity of the ε subunit for the 3'–OH terminus, which is caused by

the binding of the α subunit to the DNA56. Interaction of the ε and θ subunits slightly

stimulates the exonuclease activity as well, suggesting a possible role of θ in fidelity33.

This therefore makes an essential contribution to the network of protein-protein

interactions that stabilise the many conformational states of the replicase on the DNA.

This distinctive Pol III mechanism may allow for efficient proofreading and fast

polymerisation modes.

1.2.2 The β2 sliding clamp

The β2 sliding clamp is composed of two semicircular β monomers arranged in a

head-to-tail configuration, forming a homodimeric ring (Figure 1.3A). The association of

these monomers has a KD of 60 nM in solution. Furthermore, each β subunit is

comprised of three domains of identical topology. Due to this arrangement, the sliding

clamp has two structurally inequivalent faces (Figure 1.3), one of which contains the

two C-terminal regions from each monomer that are involved in interactions with the

other replicases, including the Pol III core. The central channel of the ring is lined by 12

α-helices and is wide enough (~34 Å diameter) to encircle and topologically bind to

dsDNA. In contrast, the outer surface is predominantly composed of β-sheets57. The

hydrogen bonded water molecules that are accommodated in between β2 and DNA

allow the clamp to freely slide along dsDNA in a sequence-independent way, enabling

highly processive interactions between the polymerases and the template. The basic

residues on the inner surface of the ring create a site of net positive electrostatic

potential that interacts with the negatively charged DNA passing through the centre32,58.

Interestingly, this passing of the DNA through the channel occurs at a pronounced

angle of 22º, which may enable switching of the DNA between the high fidelity Pol III

replicase and the low-fidelity Pol IV during lesion bypass59,60.

9

Figure 1.3: The β2 sliding clamp of E. coli DNA polymerase III holoenzyme58. A)

Front view of the crystal structure of the β2 homodimeric ring. The central channel is lined

by 12 α-helices and is large enough (~34 Å diameter) to encircle and topologically bind to

dsDNA, while the outer surface is composed of β-sheets. Each monomer is highlighted in

a different colour (red or purple), and is comprised of three domains. B) Side view

(rotated 90º around y-axis of the plane in (A)) of the crystal structure of the β2 dimer

(coloured in grey) with modelled dsDNA (coloured orange) going through the central

channel at an angle of 22º, relative to the C2 rotational axis of the clamp60. The C- and N-

terminal faces are indicated, and the hydrophobic protein-binding pockets located

between domains II and III are highlighted as yellow spheres on the C-terminal face of

both monomers.

The β2 clamp is the interaction centre for most replication proteins that manage

DNA replication and repair on dsDNA, such as the CLC, the α and ε subunits of the Pol

III core and all the other E. coli polymerases (Pols I–V)61–63. These replicases and the

DNA template are known to interchangeably bind to the clamp at different points of the

hydrophobic protein-binding pocked located between domains II and III and present on

the C-terminal face of both β monomers60. These protein–protein interactions are

possible due to the penta- or hexa-peptide CBM with the general formula QL[S/D]LxF

(where x is any amino acid, and is not always present), which are found in β2-binding

proteins and are conserved throughout eubacteria64,65. Therefore, β2 binding is

competitive and regulated in this pocket, and clamp binding proteins must be displaced

and exchanged during different phases of E. coli replication and repair in order to

sequentially bind to the same clamp66. This hierarchy of peptide binding further

suggests an ordered binding mechanism of CBM peptide recognition, which has been

recently probed using X-ray crystallography67.

10

The α–β interaction allows the core to be bound to the clamp (also referred to as

the ‘processivity factor’), which makes the replicase more efficient by significantly

increasing both the processivity (>50 kb) and the speed (~750 nt per second) of

chromosomal replication58. Thus, the β2 clamp is responsible for ensuring that the

polymerase is stably bound to the DNA template during the nucleotide incorporation

and translocation phases and, at the same time, allowing it to slide freely along dsDNA.

The β2 clamp also interacts with the δ subunit of the clamp loader complex (KD =

8 nM) through one of its two identical hydrophobic protein-binding pockets. This is also

the site where the α subunit binds, providing the basis for the switching between the

CLC and the polymerase. This δ–β2 interaction leads to the opening of the sliding

clamp by inducing a conformational change in one of the two dimer interfaces68,69. The

loading of β2 onto the primed DNA terminus is regulated by the full CLC and is

monitored by the common binding sites within the clamp (that also interact with the

DNA template)51. A proposed mechanism suggests that the interactions between the

residues in the binding pocked and the DNA template could displace the δ subunit and

hence break the CLC–β2 interaction, leading to the closing of the ring around the

dsDNA51.

The common binding pocket that recognises the conserved peptide linear motifs

of the replication proteins makes the E. coli DNA Pol III HE β subunit a potential

antibacterial drug target70,71. The protein-protein interaction hub in the sliding clamp is

essential for DNA replication and repair, and resistance to such drugs would require

many β2-binding proteins to mutate in a simultaneous fashion. Hence, inhibitor

molecules that mimic this sequential mechanism and interact at this binding site would

generate a novel class of antibiotics with low drug-resistance probability72,73.

1.2.3 The clamp loader complex

The E. coli DNA Pol III HE clamp loader complex (CLC) contains 7 subunits with

the general formula of τnγ(3–n)δδ’–ψχ (n = 0–3)74,75. The τ/γ, δ and δ’ subunits of this

complex are structurally all members of the AAA+ ATPase family and form a

pentameric ring76,87 whose main role is to perform DNA-dependent ATP hydrolysis to

load and unload the β2 sliding clamp and the Pol III core subunits onto the primed DNA

strand78,79. Hydrolysis of the ATP ejects the clamp loader from β, thereby releasing the

hydrophobic pocket in β for interaction with α.

11

The essential CLC cores involved in clamp loading are hetero-pentamer

structures in the absence of both ψ and χ subunits, where the five subunits

cooperatively bind to each other80. In the recently resolved γ31–373.δδ’ (~60 flexible

amino acid residues were cleaved from the C-terminus of each γ subunit; γ can be

substituted by τ as described in detail in Section 1.2.3.1) pentameric core structure, the

corresponding C-terminal domain III of the ATPase subunits are observed to interact in

a circular manner81,82. The asymmetrically arranged N-terminal domains I and II consist

of ATP-binding sites located at subunit interfaces that are composed of phosphate

binding loops (P-loops) in one subunit and conserved Ser-Arg-Cys (SRC) motifs in the

neighbouring one25,83. In the active state of the CLC on primer-template DNA, the ATP-

binding domains formed a helical structure around dsDNA, indicating a screw-cap-like

arrangement of the clamp loader enclosing the primed DNA84. This structure plays a

crucial role in DNA recognition, DNA-dependent CLC ATPase activity and clamp

release85.

The ψ and χ subunits strongly associate with each other in a 1:1 complex86 that

is not essential for the clamp-loading process87. However, the χ subunit does promote

polymerase processivity and β2 clamp assembly by binding to the flexible C-terminus of

SSB88,89. This interaction directs the DNA Pol III HE onto SSB-coated ssDNA90. Since

the χ subunit interacts with the CLC through the ψ subunit91, the ψ–χ complex connects

the clamp loader core to SSB. This allows the CLC to contact the primers on the ssDNA

template formed by helicase action on the lagging strand. Additionally, the χ subunit

plays a role in the primase-to-polymerase switch by out competing DnaG primase and

displacing it from RNA primers when binding to SSB bound nearby on the ssDNA

template. The competitive displacement from primase–SSB to χ–SSB recycles the

primase and enables the CLC to load the sliding clamp onto the template92. The

conserved flexible residues at the N-termini of ψ are responsible for the stable

interaction (KD = 2 nM) of the ψ–χ complex with the domain III of γ and τ93, thereby

further stabilising the CLC by increasing the affinity of τ and γ for the δδ’ complex94. The

CLC core–ψ binding also enhances its ATPase activity and affinity for the clamp and

the template.

The δ and δ’ subunits interact strongly through their respective domains III to

form a 1:1 complex68,95. This binding regulates the ability of δ to interact with β280. Once

bound to ATP, the CBM of δ interacts with the face of the β2 clamp containing the C-

12

termini during clamp loading96. A conformational change of the clamp loader is required

when loading β2 onto primed DNA. The ATP-bound state promotes structural

complementarity between the CLC and β2, which, together with the screw-cap-like

arrangement of the CLC, opens one of the dimer interfaces of the clamp before the

clamp loader positions the clamp onto the template85. The clamp opening further

stimulates the other CLC subunits such as τ/γ to weakly interact with β297.

1.2.3.1 The τ and γ subunits

The τ (71 kDa)98 and γ (47 kDa) subunits are encoded by the same dnaX gene

(Figure 1.4)99,100 and belong to the AAA+ ATPase family, conferring their ability to bind

and hydrolyse ATP101. The full length τ is composed by 643 amino acid residues

whereas γ is produced as a consequence of a –1 ribosomal frameshift during

translation of dnaX mRNA102–104. Since the N-terminal 431 residues of τ (domains I–III)

are shared with γ, either subunit can form the circular pentameric CLC core complex

with δ and δ′81. The γ subunit (431 residues) ATPase active site is in the N-terminal

domains I and II, while the C-terminal domain III is involved in oligomerisation105.

The τ subunit is arranged in five domains. The additional 24 kDa C-terminal

domains IV and V are joined to the common N-terminal fragment through a flexible

proline rich linker106. The 8 kDa domain IV (residues 430–498) is responsible for the

weak DnaB helicase binding107 while the 16 kDa domain V (residues 499–643), also

referred to as τc16 for its molecular weight, interacts tightly with the polymerase core

through the C-terminal τ-binding site of the α subunit54,108. As mentioned above in

Section 1.2.1.1, the β2 clamp also binds to the C-terminal region of the α subunit and

this competitive binding is suggested to support the dissociation of the clamp loader

from the Pol III core after its loading onto primed DNA66.

13

Figure 1.4: Domains of the τ subunit of E. coli DNA polymerase III holoenzyme54.

The full length τ is a 71 kDa protein comprised of 643 amino acid residues. The γ subunit

is produced by a –1 translational frameshift and consists of 431 N-terminal residues

identical to τ domains I–III. The N-terminal domains I and II of the τ/ γ subunits contribute

the ATPase activity, while the C-terminal domain III is involved in oligomerisation in the

CLC. The additional C-terminal fragment is joined to the common N-terminal fragment by

a flexible proline-rich linker. Domain IV (residues 430–498) is responsible for DnaB

helicase binding while the 16 kDa domain V (residues 499–643; also referred to as τC16)

interacts with the polymerase core through the α subunit. The τC16 derivative used in this

study is composed of an initial Met residue followed by the 145 C-terminal residues

(499–643). The more stable τC14 construct was formed by elimination of 18 residues from

the C-terminus and was used in structural studies; it no longer binds to α.

The DNA Pol III HE comprises two polymerase cores (when the CLC contains

two τ’s and one γ) and thus, two α subunits are responsible for the simultaneous

replication of the leading and the lagging strands109. These two subunits are connected

by the same pentameric CLC composed of γ, δ, δ′, and two τ subunits110.

Consequently, the strong interaction of α with τ within the CLC presents a critical role in

the arrangement and function of the replisome by connecting both polymerases to the

DnaB helicase.

The 3D solution structure of the C-terminal domain V of the τ subunit has been

solved by nuclear magnetic resonance (NMR) and its interaction site with the α subunit

has been mapped111. Due to the tendency of τC16 to undergo proteolysis, a more stable

truncated 14 kDa τC14 derivative (residues 499–625) was also used. Studies on τC16 also

showed the lack of structure of the flexible 26 C-terminal residues (618–643) that are

not present in the conserved folded region. Due to the closeness of the N- and C-

termini of τC16, the possible separation between the pentameric CLC and the α subunit

14

is restricted (Figure 1.5), and thus, also between the leading and lagging strand

polymerases.

Figure 1.5: Solution structure of the folded core of domain V of E.coli τ derived by

deletion of the 18 C-terminal residues from domain V (τC14)17. A superposition of 20

lowest-energy NMR conformers (green cartoons) is shown. Domain V of τ interacts

tightly (KD ~260 pM) with the C-terminal region of α54. The last 18 residues of domain V

of τ are intrinsically unstructured and are required for interaction with α. Secondary

structure prediction and mutagenesis studies suggest formation of an α-helix upon

binding to α.

The possible existence of two or three τ subunits and thus two or three Pol III

cores within the E. coli DNA Pol III HE is still being tested. Recent single molecule

studies in vivo support the potential presence of replisomes containing three α subunits

that are bound to three τ subunits in the CLC112.

The crystal structure of full length τ subunit has not yet been solved. Due to the

dynamic behaviour and flexibility of its C-terminal domains, structural analysis has been

challenging and further research is required to gain a deeper understanding of the

function and conformational changes of the DNA Pol III HE during bacterial DNA

replication.

1.3 TECHNIQUES USED FOR STUDYING PROTEIN STRUCTURE AND FUNCTION

1.3.1 DNA replication assays

DNA replication assays are used to study the enzymatic and functional

characteristics of proteins and their associated complexes such as DNA Pol III HE. The

first replication assays were developed by Arthur Kornberg and colleagues23 and were

15

challenging to perform. They utilised techniques such as quantification of incorporation

of radiolabelled mononucleotides containing 32P into DNA during synthesis or by

measuring the increase in viscosity of a solution as DNA is polymerised113,114. New

assay methodologies have made studying DNA replication significantly easier and

provided researches with a deeper understanding of the replisome. For example,

individually purified protein subunits can be assembled onto a circular template of

dsDNA in vitro and the products of continuous enzymatic synthesis around the circle

can assessed using agarose gel electrophoresis with visualisation using DNA staining.

In this project, a functional replication assay using M13 phage template DNA and

reconstituted protein subunits is utilised to study the effect on replication activity of

various mutants of the τ subunit of the Pol III HE clamp loader complex and its potential

interaction with the β2 clamp.

1.3.2 Mass spectrometry

Mass spectrometry (MS) is an analytical method that determines the molecular

weights of proteins and protein complexes and the relative abundance of molecules in a

sample. Electrospray ionization (ESI) is a ‘soft’ ionization technique in which solutions

of proteins, peptides, and other biological macromolecules are routinely acidified (for

positive ion mode) using volatile formic acid115. This procedure is widely used in mass

spectrometry to provide accurate molecular weights of denatured proteins that have

previously been purified. Proteins exposed to ESI-MS desorption method are ionized in

small droplets that are then further desolvated, which creates molecular ions with

several different charge states. The homogeneous sample is then passed into the mass

analyser through a capillary needle, resulting in a distribution of ions with mass-to-

charge ratio (m/z) values that appear as a series of peaks in a mass spectrum116. The

quantitative analysis used to calculate the molecular weight of the protein is done by

the difference between the m/z values of adjacent peaks in the spectrum, representing

sequential charge states of the ions. Calculations to determine the mass (Mr) from the

ESI-MS spectrum can be carried out by:

𝑝 = 𝑚

𝑧

𝑝1 = 𝑀𝑟 + 𝑧1

𝑧1

𝑝2 =𝑀𝑟 + (𝑧1 − 1)

𝑧1 − 1

16

where peak p1 comes before peak p2 in the spectrum, and has a lower m/z value (the

m/z value increases as the number of protons attached to the molecular ion

decreases). The z1 value represents the charge of the first peak. Ultimately, the high

sensitivity of the instrument allows the collection of extremely accurate quantitative and

qualitative measurements.

Significant advances in MS technology enable interacting proteins in non-

covalent complexes to be characterised by dissociating small molecules in the gas

phase from subunits within a macromolecular complex, providing a huge development

in the structural study of protein assemblies involved in cellular activities117,118.

1.4 AIMS OF THIS PROJECT

The crystal structure of the full length τ subunit of the CLC has not yet been

solved. However, the solution structure of the C-terminal domain V of τC14 (deletion of

18 residues from τC16) has been solved through NMR spectroscopy and the amino acid

resonances of this molecule have been assigned111. The structure of τC14 contains the

entire globular folded region of τC16 between residues Pro507 and Ser617 of domain V;

the last eight residues of τC14 were found to be mobile and the cleaved 18 residues from

τC16 were also unstructured and showed no association with the folded core. With

regard to this, the Dixon laboratory (University of Wollongong, Australia) has recently

discovered a novel interaction between domain V of the τ subunit and the β2 sliding

clamp of E. coli DNA Pol III HE (unpublished). Surface plasmon resonance (SPR)

experiments were carried out by immobilising N-terminally biotinylated version of τC24

(bio-τC24) onto a Streptavidin coated surface, and introducing a series of serially diluted

samples of the β2 clamp. These measurements showed equilibrium responses from

which a binding affinity constant of KD = 110 ± 5 M as a dimer was derived.

Subsequently, HSQC NMR analysis were performed for 15N-labelled τC14 at different β2

concentrations (with Prof G. Otting, Australian National University, unpublished). A

greater effect was seen with the τC22 derivative (containing domain IVa as well as

domain V; Figure 1.4) than with τC14. Amino acid residues with 1H chemical shift

changes >0.007 ppm were identified as a consequence of binding to β2 (Figure 1.6). As

a result, a very weak τ–β2 binding affinity of KD = 80 M was derived from these 1H

chemical shifts. Therefore, these two different techniques confirmed the same weak

17

binding affinity, which provided reliable evidence for the interaction between the β2

clamp and domain V of τ. This was an intriguing result because it suggests a new

interaction of τ with β2 may occur at some stage of replisomal dynamics during DNA

replication.

From this study, a binding interface was identified, and amino acid residues were

chosen for mutagenesis studies on full-length τ and τC16, altering positively/negatively

charged residues (R, H or D) to negatively/positively (E or K, respectively) or non-polar

aliphatic (A) residues. The residues located in the conserved globular fold of τC14 were

suggested to comprise the β2-binding site, since the chemical shifts of the ends of the

structure could be due to flexibility. Thus, the selected residues were found in this

conserved region. By testing these mutants in functional replication assays, their effects

on the process of bacterial DNA replication can be observed.

Figure 1.6: Identified amino acids in τC14 with 1H chemical shifts >0.007 ppm as a

consequence of binding to β2111. This figure highlights the residues of the τC14

structure with observable 1H chemical shift changes of >0.07 ppm in 15N-τC14. The

colours in the structure refer to the charge of the residues at the β2 binding interface: blue

indicating positively charged amino acids and red indicating negatively charged. The

amino acids chosen for mutagenesis studies of full length τ and τC16 are identified as

H562, D592 and R651 and are located in the conserved globular fold of τC14.

The ultimate goal of this project was to gain a further understanding of the

individual structures and functions of the Pol III HE subunits and their role(s) in the

essential process of DNA replication. Additionally, a deeper insight of these

mechanisms could contribute to the development of novel antibacterial drugs designed

to target proteins with low-resistance probability, such as the common binding pocket of

the β2 sliding clamp70–73.

18

2 RESULTS AND DISCUSSION

2.1 RESULTS - τ subunit and β2 sliding clamp interaction

2.1.1 Cloning of mutant genes

Initially, site-directed mutagenesis was tried to introduce charge change

mutations at His562, Asp592 and Arg651 of τC16, as indicated in Section 3.2.1.3a. After

two attempts, only wild-type τC16 clones and unintended mutants were detected by

nucleotide sequencing (Section 3.2.1.3d), so double-overlapping PCR was used

instead for the generation of the mutants.

2.1.1.1 Double overlapping PCR for generation of full-length τ mutants

Double overlapping PCR was carried out as indicated in Section 3.2.1.3c to

produce the full-length τ mutants (Figure 2.1). The dnaX+ plasmid pSJ1064 was used

as a template for PCR amplification of the gene encoding τ. Two PCR fragments I and II

were generated through incorporation of the single or double point mutation(s) using a

pair of complementary mutagenic primers and outside vector primers P9 or P10 (see

Table 1).

Figure 2.1: Agarose gel separation of double-overlapping PCR products for all full-

length τ mutants. A) Separation of fragments I and II. Lane 1: Fragment I (FI)

τR561A+H562A; Lane 2: Fragment II (F2) τR561A+H562A; Lane 3: FI τR561E; Lane 4:

FII τR561E; Lane 5: FI τH562A; Lane 6: FII τH562A; Lane 7: FI τR561E+H562A; Lane 8:

FII τR561E+H562A; Lane 9: FI τD592K; Lane 10: FII τD592K; Lane 11: FI τD592A; Lane

12: FII τD592A. B) Fragment III separation. Lane 1: τR561A+H562A; Lane 2: τR561E;

Lane 3: τH562A; Lane 4: τR561E+H562A; Lane 5: τD592K; Lane 6: τD592A. DNA

samples (3 L) were loaded onto a 1% agarose gel and run at 60 V for 40 min in TAE

buffer. The low molecular size DNA ladders (LMW; kbp) are shown on the left hand sides

of the gels.

19

Mixing of equimolar amounts of fragments I and II and using these as templates

with the outside primers P9 and P10 produced fragment III. PCR products were

separated in a 1% agarose gel as shown in Figure 2.1B.

PCR reactions showed the expected sizes of ~1.9 kbp and ~0.49 kbp for

fragments I and II, respectively. The PCR reaction where the two fragments were mixed

showed bands of ~2.2 kbp, consistent with the generation of full length dnaX fragments.

Due to time constraints, studies were more focussed on τC16 mutants rather than full

length τ, so these were left at this stage.

2.1.1.2 Double overlapping PCR for generation of τC16 mutants

The part of the dnaX gene that encodes τC16 (codons 499 to 643) was also

amplified by double overlapping PCR as described in Section 3.2.1.3c. PCR products

for all τC16 mutants were separated in a 1% agarose gel as shown in Figure 2.2.

Figure 2.2: Separation of double-overlapping PCR fragments for τC16 mutants. A)

Separation of fragments I and II. Lane 1: Fragment I (FI) τC16R561A+H562A; Lane 2:

Fragment II (F2) τC16R561A+H562A; Lanes 3 and 4: FI and FII τC16R561E; Lanes 5 and

6: FI and FII τC16H562A; Lanes 7 and 8: FI and FII τC16R561E+H562A; Lanes 9 and 10:

FI and FII τC16D592K; Lanes 11 and 12: FI and FII τC16D592A. B) Fragment III

separation. Lane 1: τC16R561A+H562A; Lane 2: τC16R561E; Lane 3: τC16H562A; Lane 4:

τC16R561E+H562A; Lane 5: τC16D592K; Lane 6: τC16D592A. The low molecular size DNA

ladders (LMW; kbp) are shown on the left hand sides of the gels. A positive control (+)

containing only wild-type τC16 was loaded onto the gel on the adjacent lane to LMW.

20

Bands from the PCR products showed the expected sizes of ~0.3 kbp for

fragment I and ~0.4 kbp for fragment II. The τC16D592K and τC16D592A mutants showed

different fragment sizes due to the distance from the restriction sites being more or less

for fragments I and II, respectively. The positive control containing only wild type τC16

template showed a single band at ~0.7 kbp. Fragment III bands presented a product of

a similar size as τC16 template at ~0.7 kbp.

PCR fragments III were isolated from agarose gels following digestion with

restriction endonucleases EcoRI and NdeI. Fragment III of the dnaX gene bearing the

τC16R561A+H562A mutations was inserted between the same restriction sites in the

temperature-inducible phage promoter vector pND706 to yield pMA2228 (Section

3.2.1.3c). Subsequently, τC16R561A+H562A was overproduced and purified using E.

coli strain AN1459/pMA2228 to examine the roles, if any, of the mutated residues in the

τC16–β2 interaction (Sections 2.1.2 and 2.1.3). Although plasmids containing different

point mutations of τC16 also directed overproduction of an ~15 kDa protein (Section

2.1.3), only the product encoded in plasmid pMA2228 showed the desired mutations of

τC16R561A+H562A.

2.1.1.3 Colony PCR on τC16 mutants in vector

Colony PCR was carried out as described in Section 3.2.1.3b to verify the

presence of inserted DNA in plasmids in transformed strains before performing

nucleotide sequencing. Transformants were grown on LB plates and 8 randomly picked

colonies were used for each mutant. PCR products for four of the τC16 mutants were

separated in 1% agarose gels as shown in Figure 2.3.

Clones picked contained the right size insert (~0.65 kbp) to encode τC16 (~16

kDa). Consequently, two positive clones for each mutant were chosen. Gene integrity of

the mutants was verified by nucleotide sequencing (Section 3.2.1.3d). Several plasmids

did not show interpretable sequences and low intensity peaks were observed. The

plasmids used for overproduction and purification of τC16 all contained the point

mutations that altered the amino acid sequence of the protein at the targeted region.

21

Figure 2.3: Agarose gel electrophoretic analysis of colony PCR for τC16 mutants in

vector. PCR product (10 L) was loaded onto 1% agarose gel and run at 70 V for 50

min in TAE buffer. A) τC16D592A; B) τC16R561E; C) τC16H562A; D) τC16R561E+H562A.

Molecular size markers (LMW) of dsDNA (size in kbp) are shown on the left of the gels.

All the visible bands are at the correct position at ~0.65 kbp.

2.1.2 Expression of τC16 mutant proteins

After cloning of the τC16 mutant gene fragments into the promoter vector

pND706 (Section 3.2.1.3c), constructed plasmids were used to direct the

overproduction of τC16 in E. coli cells. Plasmids were transformed into the strain

BL21(DE3)recA (Section 3.1.4). Inefficient transformation was observed for some of

the τC16 mutants, perhaps suggesting that background expression of the dnaX gene

might be lethal to the cells, due to binding to α subunit. τC16 mutant proteins were

expressed at high levels after a 3 h heat induction at 42ºC, where cells grew to an

OD600 of ~1.2 (Section 3.2.1.9).

The integrity of the expressed protein was verified by SDS-PAGE (Section

3.2.2.3). Proteins within these samples were separated by SDS-PAGE under reducing

conditions using 4–12% gradient gels (Bio-Rad). Coomassie blue stained SDS-PAGE

gels of whole cell lysates the τC16 mutant cells are shown in Figure 2.4.

22

Figure 2.4: SDS-PAGE gel analysis of overproduction of τC16 mutant proteins.

Cultures were harvested 3 h after induction, and samples were taken before induction.

Harvested cells were re-suspended to OD600 = 10 in equal volumes of BugBuster master

mix and 2 x loading buffer before proteins in samples (16 L) were resolved by SDS-

PAGE on 4–20% polyacrylamide gels (Bio-Rad). Gels were run at 180 V for 32 min. BI:

whole cells, before induction (growth at 30ºC); AI: whole cells after induction at 42º.

Numbers 1–3 indicate protein samples from each 1 L culture (1 L x 3). Molecular weight

markers (LMW) of protein size (kDa) are shown on the left hand side of the gel. A) SDS-

PAGE gel from the overproduction of the τC16R561A+H562A mutant. Single bands at ~

16 kDa for AI indicate successful expression of the τC16 protein. B) SDS-PAGE gel from

τc16H562A, τc16R561E+H562A and τc16D592A protein expression, respectively. Single

bands in AI lanes for each of the mutants are positioned at ~15/16 kDa. Hence, τc16

proteins containing these mutations were successfully expressed at similar size to wild

type τc16.

The position of the single bands of τC16R561A+H562A, τC16D592A, τC16H562A

and τC16R561E+H562A mutants after induction (AI) were of similar size to wild-type τC16

protein subunit at ~16 kDa. This indicated that the mutants were successfully

expressed in large scale. Each set of 3 x 1L cultures typically produced ~3 g of wet cell

pellets, and the τC16 mutants were subsequently recovered from the E. coli cells by cell

lysis and FPLC protein purification.

2.1.3 FPLC τC16 protein purification

Previously expressed τC16 proteins containing the desired mutations were

purified into isolated subunits by fast protein liquid chromatography (FPLC) as

described in Section 3.2.2.1. Purification of soluble τC16 mutant proteins was carried out

as a two-step method using a column of Toyopearl DEAE–650M anion-exchange resin

23

(Toyosoda, Japan). Prior to chromatography, a crude protein mixture was obtained

following cell lysis, protein precipitation with ammonium sulphate, resuspension of the

pellet and dialysis. In the first chromatographic step, the crude mixture was flowed

directly through the DEAE column in buffer containing 150 mM NaCl (Figure 2.5).

Under these conditions, τC16 mutant proteins flow directly through the column, but many

contaminant proteins and all nucleic acids bind and so are removed. The column is

then washed at high salt to remove the bound material so it can be reused.

Figure 2.5: FPLC purification of τC16R561A+H562A using a DEAE-650 M column:

Flow through step. The crude protein sample was previously dialysed against buffer

containing 150 mM NaCl. The DEAE-650M column was pre-equilibrated with the same

buffer. The green line indicates the buffer B % and thus the NaCl concentration. At 7.5%

buffer B (150 mM NaCl; 0–100 mL), some proteins flowed through (blue peak at ~7 first

fractions) since these did not bind to the positively charged column. Fractions 2–7

containing the desired τC16 protein were collected and pooled. Once NaCl concentration

was increased to 2 M (100% buffer B), buffer out-competed nucleic acids and other

proteins bound to the column. Blue peak at ~150 mL indicates the eluted nucleic acids

that have thus been separated from the proteins in solution.

Fractions 2–7 containing the mutated τC16 protein were collected (55 mL) after

the flow through step. Further purification steps were required to remove the

contaminant proteins that were also present in the pooled sample. It’s been shown that,

when applied to the DEAE column in lower salt buffer, τC16 binds to the resin and elutes

early in a NaCl concentration gradient as a single peak at ~30 mM NaCl. As a result,

24

this final DEAE II binding step effectively removes almost all contaminant proteins from

the isolated τC16R561A+H562A protein (Figure 2.6).

Figure 2.6: FPLC purification of τC16R561A+H562A using a DEAE-650 column:

Binding step. The sample containing the τC16 protein (Figure 2.5) was dialysed against

buffer A (0 mM NaCl). The DEAE–650M column was pre-equilibrated with the same

buffer (0% buffer B). The green line indicates the buffer B % and thus the NaCl

concentration. At 0% buffer B, many proteins bound to the column. As the NaCl

concentration was gradually increased to 9% buffer B (180 mM NaCl), the proteins

weakly bound to the column eluted first. τC16R561A+H562A elutes as a single peak at

~30 mM NaCl and fractions 33–35 under the peak were collected and pooled.

Fractions 33–35 were pooled after the final DEAE II binding step, giving to a total

of 19 mL of purified protein with a concentration of 0.87 mg mL–1. These fractions were

dialysed in storage buffer with a high glycerol concentration before storage at –80˚C.

The purity of the isolated proteins was verified by SDS-PAGE. Coomassie blue

stained SDS-PAGE gels of purified proteins are shown in Figures 2.7 and 2.8. The

τC16R561A+H562A protein was judged to be >90% homogeneous from the SDS-PAGE

gel analysis, indicating the absence of other contaminating proteins. The position on the

single bands of the τC16R561A+H562A protein subunit were consistent with the size of

the wild type τC16 protein control at ~16 kDa. The intensity of the bands showed that

fractions 33–35 contained the highest τC16R561A+H562A concentrations.

25

Figure 2.7: SDS-PAGE gel of selected FPLC fractions from the purification of

τC16R561A+H562A. A) Samples from the final DEAE step were analysed using a 4–12%

gradient SDS-PAGE gel (Bio-Rad). Fractions under the peak (30–39) in Figure 2.6

containing τC16R561A+H562A were separated on the gel. From the intensity of the bands

fractions 33–35 contained the highest τC16R561A+H562A concentration, and thus, they

were pooled for –80ºC storage. B) Pooled fractions from A (10 g of protein). Single

band showed a similar size to τC16 protein at ~16 kDa as indicated by the arrow.

Molecular weights markers (LMW) of protein size (kDa) are shown on the left hand side

of the gels.

Figure 2.8: SDS-PAGE final gel of selected FPLC fractions from the purification of

the τC16 mutants. Samples containing different τC16 mutants from the final DEAE binding

step were analysed using a 4–12% gradient SDS-PAGE gel (Bio-Rad), with 7 g of

protein loaded in each lane. Lane 1: τC16R561A+H562A; Lane 2: τC16H562A; Lane 3:

τC16R561E+H562A; Lane 4: τC16D592A. Molecular weights markers (LMW) of protein size

(kDa) are shown on the left hand side and the middle of the gel. τC16R561A+H562A

showed a similar protein size as τC16 at ~16 kDa. However, τC16H562A, τC16R561E

+H562A and τC16D592A mutants showed a slightly smaller size at ~15 kDa.

26

The τC16H562A, τC16R561E+H562A and τC16D592A mutants were also purified by

performing the two-step method using a DEAE–650M resin column. However, these

mutants presented a slightly smaller size than wild type τC16 at ~15 kDa. This qualitative

analysis could therefore indicate that those mutants had undergone proteolysis in vivo.

2.1.4 Electrospray ionisation (ESI)-mass spectrometry (MS)

To confirm the mutation of the τC16R561A+H562A protein other than by

sequencing of the gene, electrospray-ionisation mass spectrometry (ESI-MS) was

carried out by Dr. Celine Kelso to determine the accurate mass of the protein.

Approximately 30 L of protein sample was dialysed against 0.1% (v/v) of formic acid

(Section 3.2.2.5) for denaturation and protonation of the unfolded protein to occur,

which produces more accurate masses by ESI-MS.

The theoretical molecular weight was calculated from the amino acid sequence

using the Translate and ProtParam tools from the ExPASy Proteomics Server.

Molecular weights from the mass spectra were computed using MassLynx software by

assigning values of z to measured m/z values (Figure 2.9).

Figure 2.9: Electrospray-ionisation mass spectrum (ESI-MS) of the τC16R561A+

H562A protein. The protein (~30 L) was previously dialysed against 0.1% (v/v) formic

acid. Transformation of the data gave a measured mass of 16371.8 ± 0.05 Da.

27

An accurate molecular mass of 16371.8 Da for the purified τC16R561A+H562A

protein was obtained from the spectrum, which was in very good agreement with the

calculated theoretical mass of 16372.5 Da, and hence τC16R561A+H562A was verified

to contain the correct mutation.

The ESI-mass spectra of three of the other τC16 mutant proteins (τC16D592A,

τC16H562A and τC16R561E+H562A) that had been consistently purified using FPLC are

shown in Figure 2.10.

28

Figure 2.10: Electrospray ionisation mass spectra (ESI-MS) of the Δ7 τC16 mutated

proteins. The proteins (~30 L) were previously dialysed against 0.1% (v/v) formic acid.

A) τC16D592A mutant ESI-MS with a measured mass of 15654.52 ± 0.07 Da. B)

τC16H562A mutant with a measured mass of 15632.51 ± 0.08 Da. C) τC16R561E+H562A

mutant with a measured mass of 15604.09 ± 0.09 Da.

An accurate molecular mass of 15654.5 Da of the purified τC16D592A protein

was obtained from the spectrum, which did not correlate with the theoretical mass of

16479.6 Da. Instead, the measured mass was in very good agreement with the

theoretical mass of the protein once 7 amino acid residues were cleaved from the C-

terminus. This Δ7 theoretical mass of 15654.7 Da coincided with the measured mass to

within 0.2 Da. This was also true for the other two τC16 mutants. The measured mass of

the purified τC16H652A protein of 15632.5 Da concurred with the Δ7 theoretical mass of

15632.6 Da to within 0.1 Da, rather than with the theoretical mass of 16457.6 Da. The

Δ7 theoretical mass of 15605.6 Da for the τC16R561E+H562A mutant also matched with

the measured mass of 15604.1 Da; while the theoretical mass of 16430.5 Da for the full

length τC16 mutant was larger.

Therefore, it could be concluded that a C-terminal proteolysis of the τC16D592A,

τC16H562A and τC16R561E+H562A mutants had occurred in vivo. The cleaved 7 amino

acid residues (Δ7) showed a sequence of –EESIRPI.

29

2.1.5 Functional DNA replication assay

A rolling-circle replication assay was carried out with Dr. Slobodan Jergic to

investigate the effect of τC16R561A+H562A mutant on bacterial DNA replication in vitro.

The template used in this assay was a 5'-flap primed circular M13 template [5'-flap ss;

36-nt non-complementary 5'-flap and 33-nt complementary to wild-type circular M13

ssDNA template] (Section 3.2.3). The products were separated on an agarose gel and

stained with a dye that detects both ss and dsDNA. The proteins are first assembled on

the 5’-flap-primer, which mimics the DNA structure of a replication fork during DNA

replication. DNA synthesis occurs at the 3' end of the primer (complementary to M13) to

convert the ssDNA template (ss) to tailed-form II (TF II) by the Pol III HE in the

presence of SSB; no helicase was present to unwind the replication fork to enable the

continuous leading-strand synthesis. Strand-displacement synthesis was observed to

work best at high dNTP concentrations, and required the Pol III core (αεθ), the clamp

loader complex (γ3δδ’), the sliding clamp (β2) and SSB. Therefore, it presents a good

system to test the functionality of the τC16 subunit.

The 5’-flap primed circular M13 ssDNA (~3.0 kbp) template migrates in the gel

similarly to a 3.0 kbp linear dsDNA marker. Lane 1 in Figure 2.11 shows a long product

arising from the expected strand-displacement (SD) DNA synthesis when no τC16 is

added to the reaction. In the presence of wild type τC16 replication is inhibited (Lane 2),

while in previous studies it has been observed that τC24 stimulates replication. Other τC16

mutants that had been previously purified by Dr. Jergic were also used to determine

their properties in this assay56. A qualitative analysis of the gel defines how efficient

(inhibition of) replication is for each of these τC16 mutants. DNA products that are longer

on average indicate less inhibition by the mutant, and thus, τC16R561A+H562A mutant

(Lane 3 and 9) is compared to wild type and mutated τC16 (Lanes 4–9). The τC16I618T,

τC16D636G and τC16L635P mutants showed a moderate effect on replication. The

τC16S617P and τC16F631I mutations had intermediate effects, while the major effect was

observed for the τC16L627P mutant.

30

Figure 2.11: Pol III strand displacement (SD) rolling-circle DNA synthesis assay. A)

Schematic of the rolling circle replication assay. M13 5’-flap-ss template is firstly

replicated to a tailed form II (TF II) by the Pol III HE in the presence of SSB and then

strand-displacement synthesis proceeds further at the fork (continuously synthesised

DNA is represented by curved line). B) Agarose gel electrophoresis analysis of rolling-

circle replication strand-displacement assay with wild type τC16 and different τC16 mutants.

Lane 1: Primed M13 ss; Lane 2: wild-type τC16; Lane 3: τC16R561A+H562A; Lane 4:

τC16S617P; Lane 5: τC16I618T; Lane 6: τC16L627P; Lane 7: τC16F631I; Lane 8: τC16L635P;

Lane 9: τC16D636G; Lane 10: τC16R551A+H652A. The dsDNA size markers (LMW; kbp)

are shown on the left and right hand sides and selected marker sizes are listed on the

left of the gel. Simplified DNA products as explained in A) are drawn on the left hand

side of the Figure.

The τC16R561A+H562A double point mutation did not disturb replication

noticeably compared to wild type, having a much smaller effect than some other

mutations in τc16 (Figure 2.11). However, some lower level of inhibition by

τC16R561A+H562A compared to τC16 wild type could be observed, but the difference

was still relatively minor to assign big conclusions. Perhaps a greater functional effect

will be observed with the mutants that are modified to a residue with the opposite

charge rather than a neutral aliphatic residue.

31

2.2 DISCUSSION

2.2.1 Overproduction and purification of τC16 proteins

Overproduction and purification of non-native subunits of the replisome is often a

challenging process since most of these individual proteins are lethal to the cell. The

τC16 protein has been determined to be toxic for the E. coli cell. This is suggested to be

caused by its affinity to bind to the α subunit, which competes with native τ in the full

clamp loader complex54. Hence, this sequestration of α will affect the efficiency of

replication. Toxicity was suggested by low transformation efficiencies of the constructed

plasmids into the E. coli strains AN1459 and BL21(DE3)recA. To improve host viability

during target transformation and protein expression, the τC16 coding region was sub-

cloned into a highly-regulated phage promoter vector. This system relies on use of a

temperature-sensitive repressor, cI857ts, which is also expressed from the cloning

vector. When cells are grown at 30˚C, the repressor is active and prevents transcription

from powerful tandem promoters. On shift to growth at 42˚C, the repressor denatures

in vivo, and high-level transcription is turned on.

Nevertheless, even under these highly-regulated conditions, the other τC16

mutants (τC16D592A, τC16H562A and τC16R561E+H562A) underwent proteolysis in vivo

to yield a final species with a molecular weight of ~15 kDa, as assessed by SDS-PAGE

and ESI-MS. These measurements indicated that these mutants lacked 7 amino acid

residues from their C-termini. Removal of the last 18 residues of τC16 (τC14) has been

shown to suppress its lethal phenotype, which provides evidence for the interaction

between α and the C-terminal residues in τ54. Additionally, the same study showed that

τC16Δ7 deletion mutants did not form a stable complex with α. Therefore, τC16Δ7

proteolysis could present a more stable mutated product that is less toxic for E. coli due

to reduced binding affinity to α through its C-terminal domain V. However, a promoter

vector was also used to prepare these mutant proteins due to low level of expression

observed on the genes encoding τC16Δ7. Although it is difficult to identify the step in

which the deletion of 7 amino acids occurred in vivo, these mutants resulted in soluble

proteins that could be purified readily. Further lethality studies need to be carried out to

determine the action of these mutants and the effectiveness of their binding to α.

32

Following overproduction using plasmid pMA2228, τC16R561A+H562A was

purified in a yield of 19 mg from 3 litres of cell culture (Section 2.1.3). Despite the

relatively large amounts of liquid culture used for cell growth and protein expression,

yields of these proteins were generally satisfactory after the final purification step. Done

thoroughly, the FPLC two-step method used ensures the removal of DNA and any

remaining soluble material after cell lysis and leaves the τC16 mutated proteins in a

highly purified form (Section 3.2.2.1). The purity of the isolated subunit was assessed

by SDS-PAGE, where single bands in the gel indicated the absence of contaminating

proteins. Accurate molecular weights determined by ESI-MS verified that the domain V

fragment contained the amino acid residues mutated in the desired sites of R561A and

H562A.

The successful homogeneous purification of the τC16R561A+H562A protein was

an essential step in order to perform in vitro functional replication assays, where

contaminants could interfere with protein assembly and functionality.

2.2.2 Functional effects of τC16R561A+H562A protein

The successfully purified τC16R561A+H562A protein presented the 16.372 kDa

subunit of the clamp loader complex that contained mutations of the residues that are

prone to interact with the β2 sliding clamp. Functionality of the τC16R561A+H562A

double point mutations was measured in vitro in a rolling-circle replication assay

performed by Dr. Slobodan Jergic (Section 2.1.5). In this study, τC16R561A+H562A was

found to not disturb bacterial DNA replication noticeably. Inhibition of replication has

been extensively observed when adding wild type τC16 to a strand displacement DNA

replication assay. The reason for this effect in replication efficiency when τC16 is present

has not yet been fully understood and experimental evidence will be needed to attain a

deeper insight. However, inhibition is suggested to be caused by the binding of τC16 to

the α subunit of the Pol III core, and thus destabilising α onto DNA. Previous

mutagenesis studies have revealed that the binding interface of τ–α interaction possibly

comprises the highly conserved last C-terminal 18 residues of τC16; while it has also

been observed that τC24 stimulates replication, possibly due to its reduced affinity for the

C-terminal region of α.

33

Qualitative analysis of the strand-displacement assay gel using wild type τC16 and

different τC16 mutants assessed the inhibition level caused by these proteins. A longer

DNA product indicated a lower level of inhibition by the τC16 mutants, and thus a weaker

binding to the α subunit. Prior examinations of the single point mutants synthesised by

Dr. Slobodan Jergic revealed that no single amino acid mutation was sufficient to

completely disrupt the τ–α interaction, presumably because the interaction interface is

comprised of residues located in a flexible region of τ54.

When testing how the mutations within the putative β2-binding site in τC16 affect

SD synthesis, τC16R561A+H562A showed a minor difference compared to wild type

τC16. Therefore, no considerable conclusions about the functionality of τC16 can be

drawn from this assay. However, the slightly more efficient replication observed in the

gel by the τC16R561A+H562A mutant suggests that it binds more weakly to the α

subunit. In contrast, this inhibition could be caused by other interactions and

functionality effects of the τC16 subunit, such as binding to the β2 sliding clamp. Whether

this reduced inhibition is due to the disrupted binding to α or β2 remains to be shown.

Subsequent studies employing the τC16 proteins containing mutations to acidic/basic

residues could present a greater effect in bacterial replication and protein binding.

Overall, this bulk assay does not provide significant information about the

possible τ–β2 interaction, and further research using the full clamp loader complex will

be needed to determine the functional effect of the mutations in the putative clamp-

binding site in the τ subunit.

34

3 EXPERIMENTAL

3.1 GENERAL MATERIALS

3.1.1 Oligonucleotide primers

Oligonucleotide primers (sequences in Table 1) were obtained from GeneWorks

(Hindmarsh, Australia). Oligonucleotide primers were dissolved in TE buffer (10 mM

Tris.HCl, pH 8.0, 1 mM EDTA) to a stock concentration of 100 M and stored at –20C.

Table 1: Oligonucleotides used in mutagenesis of τC16.

3.1.2 Buffers

Buffers used were: Lysis buffer (50 mM Tris.HCl, pH 7.6, 1 mM EDTA, 1 mM

dithiothreitol (DTT), 20 mM spermidine); buffer A (50 mM Tris.HCl, pH 7.6, 2 mM DTT,

1 mM EDTA, 5% (v/v) glycerol); buffer B (50 mM Tris.HCl, pH 7.6, 2mM DTT, 1 mM

EDTA, 5% (v/v) glycerol, 2M NaCl); dialysis buffer (50 mM Tris.HCl, pH 7.6, 2 mM DTT,

1mM EDTA, 5% (v/v) glycerol, 150 mM NaCl); storage buffer (50 mM Tris.HCl, pH 7.6,

3mM DTT, 1 mM EDTA, 100 mM NaCl, 20% (v/v) glycerol); MS buffer (0.1% formic

acid); TE buffer (10 mM Tris.HCl, pH 7.6, 1 mM EDTA); TAE buffer (40 mM Tris.HCl,

pH 7.6, 20 mM acetic acid, 1 mM EDTA); gel loading buffer (0.2% (w/v) bromophenol

blue, 50% (w/v) glycerol, 50 mM DTT and 3% (w/v) SDS in water); gel running buffer

Primers

Oligonucleotide sequences

τC16R561E (F)

5'-GCATTTGCGCTCCTCTCAGGAACATTTGAACAACCGCGGTG-3'

τC16R561E (R)

5'-CACCGCGGTTGTTCAAATGTTCCTGAGAGGAGCGCAAATGC-3'

τC16H562A (F)

5'-GCGCTCCTCTCAGCGGGCGTTGAACAACCGCGGTG-3'

τC16H562A (R)

5'-CACCGCGGTTGTTCAACGCCCGCTGAGAGGAGCGC-3'

τC16R561E+H562A

(F) 5'-TCTGCATTTGCGCTCCTCTCAGGAAGCGTTGAACAACCGCGGTGCACAGC-3'

τC16R561E+H562A

(R) 5'-GCTGTGCACCGCGGTTGTTCAACGCTTCCTGAGAGGAGCGCAAATGCAGA-3'

τC16D592K (F)

5'-TTGAACTGACTATCGTTGAAGATAAAAATCCCGCGGTGCGT-3'

τC16D592K (R)

5'-ACGCACCGCGGGATTTTTATCTTCAACGATAGTCAGTTCAA-3'

τC16D592A (F)

5'-TTGAACTGACTATCGTTGAAGATGCGAATCCCGCGGTGCGT-3'

τC16D592K (R)

5'-ACGCACCGCGGGATTCGCATCTTAACGATAGTCAGTTCAA-3'

τC16R561A+H562A

(F) 5'-CTGCATTTGCGCTCCTCTCAGGCCGCGTTGAACAACCGCGGTGCACAG-3'

τC16R561A+H562A

(R) 5'-CTGTGCACCGCGGTTGTTCAACGCGGCCTGAGAGGAGCGCAAATGCAG-3'

P9

5'-GGCAGCATTCAAAGCAGAAG-3'

P10

5'-GTTGGGTAACGCCAGGG-3' Sequences generating τC16 mutations are underlined.

F= Forward primer; R = Reverse primer.

35

(192 mM glycine, 25 mM Tris, 0.1% (w/v) SDS in water, pH 8.3); replication buffer (25

mM Tris.HCl pH 7.6, 10 mM MgCl2, 10 mM DTT and 130 mM NaCl)

MilliQ water [18.2 MΩ cm–1 resistivity] (Millipore, USA) was used in all

experiments and was sterilized by autoclaving at 121C for 20 min for molecular biology

and protein biochemistry experiments. The purity of all chemicals and reagents were of

at least molecular biology grade. All buffers were filtered through a 0.22 m filter.

3.1.3. Chemicals and reagents

Table 2: List of the chemicals and reagents used.

Supplier Chemicals and reagents

Ajax Finechem, acetic acid

Australia ammonium sulfate [(NH4)2SO4]

ethanol

36% hydrochloric acid (HCl)

propan-2-ol (isopropanol)

magnesium chloride (MgCl2)

methanol

AMRESCO, bromophenol blue

USA glycerol

glycine

sodium dodecyl sulfate (SDS)

coomassie brilliant blue

Astral Scientific, dithiothreitol (DTT)

Australia ethidium bromide (EtBr; 10 mg mL-1 in water)

Bacto Laboratories, Bacto tryptone

Australia Bacto yeast extract

Difco agar technical

Difco Luria-Bertani (LB) broth, Miller

Bio-Rad, Precision Plus Protein Dual Color standards (protein size markers)

Australia

Bioline, 2'-deoxyadenosine-5'-triphosphate (dATP), lithium salt; 100 mM solution

Australia 2'-deoxycytidine-5'-triphosphate (dCTP), lithium salt; 100 mM solution

2'-deoxyguanosine-5'-triphosphate (dGTP), lithium salt; 100 mM solution

2'-deoxythymidine-5'-triphosphate (dTTP), lithium salt; 100 mM solution

HyperPAGE (protein size markers)

Fluka, cesium iodide (CsI)

USA dimethyl sulfoxide (DMSO)

formic acid

phenylmethanesulfonyl fluoride (PMSF)

sucrose

Roche Diagnostics, ampicillin

Switzerland complete protease inhibitor cocktail tablets

36

Sigma-Aldrich, 2-amino-2-hydroxymethyl-propane-1,3-diol (Trizma/Tris)

USA 2,2',2'',2'''- (ethane-1,2-diyldinitrilo) tetraacetic acid (EDTA)

spermidine

thymine

xylene cyanol FF

sodium chloride (NaCl)

sodium hydroxide (NaOH)

Thermo Fisher Scientific, GeneRuler 1 kb DNA Ladder, 250-10,000 bp (DNA size markers)

Australia GeneRuler DNA Ladder Mix (DNA size markers)

3.1.4 Bacterial strains

The E. coli K-12 strain AN1459 (F‾ ilvC leuB6 thr-1 supE hsdR recA srlA::Tn10)

was used as a host strain for the propagation, manipulation and characterization of

recombinant DNA plasmid119.

The strain BL21(DE3)recA is a derivative of the E. coli B strain BL21 [F‾ ompT

(lon) hsdSB (rB‾ mB‾) recA srlA::Tn10], which contains a prophage carrying the T7

RNA polymerase gene under control of lacUV5 promoter/operator. BL21(DE3) is used

as an expression host to overproduce recombinant proteins. This strain is not typically

used for production of toxic proteins. The introduction of a chromosomal recA mutation

increases the stability of the inserts by preventing homologous recombination120,121.

3.1.5 Plasmid vectors

The pND706122 vector was used to carry inserts containing the mutations to

construct all the plasmids in the experiments of this project. The physical map of this

vector is shown in Figure 3.1 and the individual characteristics are described below.

37

Figure 3.1: The plasmid map of the parental plasmid vector pND706 used to

construct C16 mutant genes. RBS = ribosome-binding site (orange); MCS = multiple

cloning site (green); bla = ampicillin resistance gene; ori = ColE1 replicon. P9 and P10

indicate primer sites used for colony PCR and nucleotide sequence determination. τC16

mutant genes were inserted between restriction sites NdeI and EcoRI so as to place

them under control of the strong tandem pR and pL promoters from bacteriophage

(blue). Temperature sensitive repressor clts857 results in derepression of both

promoters by rapid temperature shift to, and maintenance at 42C.

The genes of interest are inserted into the multiple cloning site (MCS) region of

the vector that is located downstream to the strong tandem pR and pL promoters from

bacteriophage and a ribosome-binding site (RBS) to stimulate the expression of the

insert. The vector contains a unique NdeI site (5'-CATATG) containing an ATG initiation

codon allowing for in-frame target gene expression. The vector also contains a colE1

origin of replication (ori) and a bla (β-lactamase) gene that provides ampicillin

resistance to the host strain carrying the vector.

Transcription of the inserted gene is regulated by tandem pR and pL operators

and prevented by a thermolabile mutant of the repressor (product of the clts857

gene). The repressor blocks transcription when the cells containing derivatives of

pND706 are grown at 30C. Thermal induction by increasing the temperature to 42C

results in derepression of both pR and pL promoters, allowing transcription of the

38

inserted gene. Gene expression regulated by tandem promoters pR and pL is tightly

controlled, thus allowing toxic proteins like C16 to be successfully overproduced in E.

coli. Moreover, the presence of the par region from pSC101 enhances plasmid stability

at a high-copy-number in the absence of ampicillin selection.

3.1.6 Growth media

3.1.6.2 Lysogeny broth liquid medium

LB culture medium supplemented with 25 mg L–1 thymine (LBT) and 100 mg

mL–1 ampicillin (LBTA), was used to grow E. coli AN1459 and BL21(DE3)recA strains

containing plasmids. LBT media were autoclaved and supplemented with 100 mg mL–1

ampicillin (0.22 m filter sterilized) before culturing cells. All bacterial strains were

grown in broth cultures at 30C to inhibit C16 expression. One loop of cells was

collected from agar plates containing τC16 mutants and used to inoculate one litre of

LBT medium.

3.1.6.1 Lysogeny broth solid medium (agar plates)

All cells used carrying pND706 and were grown on lysogeny broth (LB)

medium123 agar plates (15 g L–1 agar in LB medium with 100 g mL–1 ampicillin) in an

incubator (Thermo Scientific, Australia) at 30C.

3.2 METHODS

Commercially available kits described in this section were used as described in

the manufacturers’ manuals, unless otherwise indicated.

3.2.1 Molecular biology methods

3.2.1.1 Preparation of plasmid DNA by alkaline lysis

Small-scale plasmid preparations (Mini-prep) were carried from E. coli cells

containing the plasmid of interest grown overnight at 30C on LBTA plates and lysed

using a commercially available QIAprep Spin Miniprerp Kit (Qiagen, USA). Extracted

plasmid DNA was eluted in warm TE buffer and stored at –20C. Plasmid DNA

extracted in this way was used for transformation of competent cells and analysis of

constructed plasmids.

39

3.2.1.2 Agarose gel electrophoresis

A Mini-Sub Cell GT agarose gel electrophoresis system (Bio-Rad) was used to

analyse the size and purity of DNA. Sizes of DNA fragments were estimated by

comparison with the DNA size marker DNA HyperLadder™ I (Bioline). Either 0.75 or

1%, (w/v) of agarose gel in 1 x TAE buffer containing 0.5 g mL–1 ethidium bromide

(EtBr) were set using the Bio-Rad apparatus based on the target size of DNA. The

same 1 x TAE buffer containing 0.5 g mL–1 EtBr was used as running buffer for the

electrophoresis. Samples were loaded with 6 x DNA loading dye (LD; 0.25%

bromophenol blue, 40% (w/v) sucrose)124. Electrophoresis was typically run between

60–80 V potential difference controlled by a PowerPac Basic power supply (Bio-Rad)

until appropriate resolution of bands had been obtained. The results were visualized

under 302-nm UV light and recorded by a GelDoc XR UV transilluminator controlled by

Quantity One® v.4.6.9 software (Bio-Rad).

3.2.1.3 Polymerase chain reactions (PCR)

All PCR experiments were performed using a DNA Engine C1000™ Thermal

Cycler with dual 48/48 fast reaction module (Bio-Rad). 200 L thin-wall flat cap PCR

tubes were used to carry out all reactions. Polymerases with their own supplied reaction

buffers (KOD DNA polymerase from Novagen, Germany; PfuTurbo DNA polymerase

from Stratagene, USA; BIOTAQ Red DNA polymerase from Bioline, Australia; Velocity

from Bioline, Australia) were used in different types of PCR described below.

Deoxynucleotides (dNTPs) stock (final concentration 25 mM each) were made up by

mixing equal volumes of each (100 mM dATP, dCTP, dTTP, and dGTP; Bioline).

3.2.1.3a Site-directed mutagenesis (SDM) PCR

The PCR protocol for SDM was based on the QuickChange site-directed

mutagenesis kit (Stratagene; QuickChange® Site-Directed Mutagenesis Kit: Instruction

Manual, 2006). The reaction cycles used to amplify the entire plasmid were the

following: a single 1 min pre-denaturation step at 95C followed by 18 PCR cycles of a

30 s denaturation at 95C, 30 s annealing at 55C, and 9 min polymerization at 68C.

This was followed by a final 12 min extension step at 68C. After SDM PCR, DpnI

digestion was carried out at 37C for 2 h to selectively remove wild-type (GmATC;

40

adenosine-methylated) template (Section 3.2.1.5). The digested DNA products were

subsequently transformed into E. coli strain AN1459 (Section 3.2.1.8).

3.2.1.3b Colony PCR

Colony PCRs were used to screen for positive clones after ligation of PCR

products into pND706. Reaction mixtures (20 L) contained 0.3 M of each P9 and P10

primers, 0.2 mM of each dNTP, 1.25 units of BIOTAQ Red DNA polymerase (Bioline,

Australia), 1.5 mM MgCl2, and 1 x PCR buffer. Single colonies were picked and washed

in MilliQ water (50 L each) and 1 L was added to the reaction mixtures. Following

this, a PCR reaction cycle containing a single 2 min pre-denaturation step at 94C

followed by 31 PCR cycles of a 30 s denaturation at 94C, 30 s annealing at 55C, and

90 s polymerization at 72C, followed by a final 10 min extension step at 72C. To

identify positive clones, the reaction mix was loaded directly onto a 1% agarose gel for

electrophoresis (Section 3.2.1.2) and DNA fragments were compared to the expected

sizes using DNA standards from HyperLadder™ I (Bioline, Australia).

3.2.1.3c Double-strand overlap PCR

To construct both single and double point mutants of τC16 by double strand

overlap PCR, pSJ1064 was used as a template54. Either the 3' or the 5' portion of the

gene was amplified using a mutagenic primer (Table 1) and one of the outside primers

P9 or P10 (Table 1). For example to construct the τC16R561A+H562A mutant gene, a

PCR reaction containing a 5’ fragment containing 136 bp upstream of the NdeI site of

the start codon and the double point mutation R561A+H562A were incorporated by

using primers P9 and τC16R561A+H562A R, yielding Fragment I. A second PCR used

primers τC16R561A+H562A F and P10 to generate Fragment II, incorporating the same

mutations and extending 110 bp past the TGA stop codon and EcoRI site. Equimolar

amounts of isolated Fragments I and II were then used as templates for PCR with the

outside primers P9 and P10 to generate a product that was then digested with NdeI and

EcoRI and isolated from a gel (Section 3.2.1.5) to yield Fragment III. Fragment III was

ligated in 3:1 molar ratio with the 4315 bp NdeI–EcoRI fragment of pND706122, to

generate pMA2228 that directs overproduction of τC16R561A+H562A.

41

Figure 3.2: Cloning strategy used to generate τC16 mutants. First 5’ and 3’ fragments

of the gene were amplified by PCR to yield Fragments I and II using a mutagenic primer

and either P9 or P10. Fragments I and II were then mixed at equimolar ratio and

amplified by PCR using primers P9 and P10 to generate Fragment III, a continuous gene

product that now contains mutation(s). Fragment III is then digested with NdeI and EcoRI

and ligated into pND706.

3.2.1.3d Nucleotide sequence determination

The nucleotide sequences of the genes of interest from cloning experiments

were confirmed by nucleotide sequence determination. PCR reactions were performed

using the BigDye Terminator (BDT) v3.1 Cycle Sequencing Kit (Applied Biosystems,

USA) with either a forward primer (P9) or a reverse primer (P10). These primers anneal

42

in regions flanking the MCS of pND706125 and their oligonucleotide sequences are

listed in Table 1. Reaction mixtures (20 L) contained BigDye sequencing buffer, 5 M

of either forward or reverse primer, ~250 ng of plasmid template and 1 x BDT mix.

The PCR reaction cycle conditions were: a single pre-denaturing step at 96C for

1 min, followed by 15 PCR cycles of a 10 s denaturation at 96C, 5 s annealing at

50C, and 75 s polymerization at 60C; 4 PCR cycles of 10 s denaturation at 96C, 5 s

annealing at 50C, and 90 s polymerization at 60C; and 4 cycles of 10 s denaturation

at 96C, 5 s annealing at 50C, and 2 min polymerization at 60C.

After PCR reactions, BDT and excess fluorescent ddNTPs were removed using

the DyeEx 2.0 spin kit (Qiagen, USA). The eluate was dried in a Savant SpeedVac

SC110 concentrator equipped with a RF40-11 rotor (Thermo Scientific) for ~30 min and

then submitted to the sequencing facility at the School of Biological Sciences,

University of Wollongong for analysis using a 3130xI Genetic Analyzer (Applied

Biosystems). Chromatograms were base-called using the program Sequencing

Analysis 5.2 (Applied Biosystems) to obtain the nucleotide sequences and the resulting

sequences were assembled using ChromasPro software

(http://technelysium.com.au/wp/chromaspro/) with target sequences modified from

UnitProtKB (http://www.uniprot.org/) to contain the correct mutation(s).

3.2.1.4 Quantification of DNA

The concentrations of purified plasmids were quantified spectrophotometrically

at 260 nm, assuming A260 = 1.0 corresponds to 50 g mL–1 dsDNA using a NanoDrop

2000c spectrophotometer (Thermo Scientific). The purity of DNA was judged by the

ratio of A260/A280, where pure DNA has a ratio of 1.8–2.0, and protein-contaminated

DNA has a ratio <1.8. The quantity of vector and insert DNA fragments separated by

agarose gel electrophoresis (Section 3.2.1.2) was also estimated based on the

fluorescence intensity from the EtBr-bound DNA compared with the DNA standard

HyperLadder™ I (Bioline). This enabled calculation of relative amount of vector and

insert to be mixed for ligation reactions (Section 3.2.1.7).

3.2.1.5 Restriction endonuclease digestion

Restriction endonucleases NdeI and EcoRI, and buffers were supplied by New

England BioLabs (USA). Restriction digestion reactions (50 L) containing 20 units

43

each of NdeI and EcoRI in CutSmart buffer were carried out at 37C for 3 h. The

reactions were terminated by heat inactivation of restriction endonucleases at 65C for

20 min. Products of the digestion were analysed and isolated following agarose gel

electrophoresis (Section 3.2.1.6).

3.2.1.6 Purification of DNA by extraction from agarose gels

DNA fragments from restriction endonuclease digestion that had been separated

in an agarose gel containing EtBr were visualized under low-energy UV light (>320 nm)

from a bench-top UV lamp and identified by comparison with the DNA standard

HyperLadder™ I (Bioline) (Section 3.2.1.2). The bands of interest were cut out using a

sterile scalpel blade. DNA was extracted from the gel using the QIAquick Gel Extraction

kit (Qiagen, USA). Extracted DNA samples were quantified as described in Section

3.2.1.4.

3.2.1.7 DNA ligation

Vectors and inserts were ligated by T4 DNA ligase (2 U; Thermo Scientific,

USA). A consistent 1:3 molar ratio of vector to insert was used for all ligation reactions.

This mixture (20 L) was incubated at ~6C overnight (~18 h) in a cold room.

3.2.1.8 Transformation of plasmids into competent E. coli strains

Competent E. coli cells were chemically transformed with plasmid DNA126.

Briefly, 7 L of the ligation mixture was transformed into 100 L of chemically-

competent E. coli strain AN1459. The cells were then cooled in ice for 30 min, followed

by heat-shocking at 30C for 2 min, and finally cooled on ice for 5 min. Cells were

recovered in 500 L of non-selective LBT medium and allowed to incubate for 1 h at

30C with shaking. The suspension was then spread on LBT agar plates supplemented

with 100 g mL–1 ampicillin and grown overnight at 30C. Resulting single transformant

colonies were picked for screening using colony PCR (Section 3.2.1.3b).

3.2.1.9 Overproduction of τC16 proteins

E. coli strain BL21(DE3)recA carrying plasmids containing C16 mutants (e.g.

pMA2228) were grown at 30C in LBT medium containing 100 mg L–1 ampicillin to A600

= 0.7. To induce overproduction, the temperature was rapidly increased to 42C. After a

44

further 3 h at 42C, and then chilled in ice. Cells were harvested by centrifugation

(16900 × g; 10 min), frozen in liquid N2 and stored at –80C.

3.2.2 Protein biochemistry

3.2.2.1 Fast protein liquid chromatography (FPLC) purification of proteins

All protein purifications and manipulations were carried out at 6°C in a dedicated

cold room. The ÄKTA systems (ÄKTApurifier), equipped with fraction collector Frac-

920, were controlled by UNICORN v5.11 software (GE Healthcare, Sweden), which

allows real-time manual control or programmed methods control and evaluation for

protein purification. All chromatography columns and lines were washed prior to protein

sample loading with MilliQ and then the appropriate buffer. Protein elution from

attached columns was monitored by absorbance at 280 nm (A280) for protein, and

conductivity. The composition of collected protein fractions was consistently checked by

SDS-PAGE (Section 3.2.2.3) analysis for purity.

After thawing, cells (2.5–3.5 g from 3 L of culture) were resuspended in 30 mL of

lysis buffer and two protease inhibitor tablets (Roche, Switzerland) and 0.7 mM PMSF

were added to inhibit proteolysis. The cells were lysed by being passed twice though a

French press (12000 psi); cell debris were then removed from the lysate by

centrifugation (35000 × g; 20 min) at 6C using a Sorvall RC 6+ centrifuge (Thermo

Fisher Scientific) equipped with a SS-34 rotor (Thermo Fisher) to yield soluble fraction I.

Proteins in fraction I were fractionated by addition of solid ammonium sulfate (0.4 g mL–

1) and stirring for 60 min. Precipitated proteins were collected by centrifugation (35000

× g; 40 min) and dissolved in buffer A+150 mM NaCl (35 mL). The solution was

dialysed against 2 L of the same buffer for 3 h to yield fraction II. Fraction II was applied

by gravity to a column (2.5 x 10 cm) of Toyopearl DEAE-650M resin that had been

equilibrated in buffer A+150 mM NaCl. Fractions containing C16 mutants that did not

bind to the column were pooled and dialysed against 2 L of buffer A overnight. The

dialysate (fraction III, 50 mL) was loaded by gravity onto a column (2.5 x 10 cm) of the

same resin, now equilibrated in buffer A. The column was washed with 100 mL of buffer

A, and τc16 was eluted in a linear gradient of 0–180 mM NaCl over 600 mL in buffer A.

All C16 mutant proteins eluted in a single peak at about 30 mM NaCl. Fractions

45

containing τc16 mutants were pooled and dialyzed against 2 L of storage buffer overnight

to give fraction IV. Aliquots were frozen in liquid N2 and stored at –80C.

3.2.2.2 Dialysis of proteins

Dialysis was performed to exchange buffer components during protein

purification (Section 3.2.2.1) and mass spectrometry measurements (Section 3.2.2.5).

Spectra/Por Standard RC (regenerated cellulose) dialysis tubing (Spectrum

Laboratories, USA) was used for dialysis of proteins. An appropriate molecular-weight-

cut-off (MWCO) of the dialysis membrane was selected to be smaller than the

molecular weight of the proteins of interest (typically 6–8 kDa). Dialysis was carried out

in 2 L of buffer for a minimum of 3 h in a cold room at 6C.

3.2.2.3 Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis

(PAGE)

SDS-PAGE was used to identify the protein content in samples overproduced in

host strains (Section 3.2.1.9) or fractions collected during protein purification (Section

3.2.2.1). Bio-Rad Miniprotean TGX pre-cast polyacrylamide gels (4–20% gradient) were

used in all experiments. Gels were run using a Bio-Rad Mini-PROTEAN tetra cell gel

apparatus. The protocol used was based on that described by Laemmli (1970)127. The

running buffer for gels was 192 mM glycine, 25 mM Tris, 0.1% (w/v) SDS in water, pH

8.3. Molecular weight markers used for SDS-PAGE gels were Dual Precision Plus

Protein (Bio-Rad). Protein samples (16 L) were mixed 1:1 with 2 × gel loading buffer

(0.2% (w/v) bromophenol blue, 50% (w/v) glycerol, 50 mM DTT and 3% (w/v) SDS in

water). Gel electrophoresis was controlled by a PowerPac Basic power supply (Bio-

Rad) and were typically run at 180 V for 32 min, or until the dye front had reached the

bottom of the gel. Gels were stained by microwave-heated staining solution [0.2% (w/v)

Coomassie brilliant blue, 40% (v/v) methanol, 10% (v/v) acetic acid] for at least 10 min

under gentle shaking and then destained by microwave-heated destaining solution

[40% (v/v) propan-2-ol and 10% (v/v) acetic acid] until the background stain was clear

under gentle shaking. The gel results were captured by scanning.

3.2.2.4 Quantification of proteins

The UV absorbance of proteins at 280 nm (A280) was directly measured using a

NanoDrop 2000c (Thermo Scientific). Molar extinction coefficients of proteins at 280 nm

46

were calculated according to the ExPASy Proteomics Server (ExPASy – ProtParam

tool; http://web.expasy.org/protparam). Since reducing agent was always present in the

buffer, no oxidised cysteine (disulfide linkage between two cysteine residues) were

likely formed in the protein sample, so the number of cysteine residues was not

considered to contribute to the molar extinction coefficients of a particular protein128.

3.2.2.5 Electrospray ionization (ESI)-mass spectrometry (MS)

The accurate molecular mass of purified proteins was routinely analysed after

purification using electrospray-ionisation mass spectrometry (ESI-MS), which can be

accurate to <0.01% of the total molecular mass of the protein (i.e. 1 Da error for a

protein of 10,000 Da). This technique provides another means to estimate protein purity

and determine the accurate mass of purified proteins. Mass spectra were acquired

using a quadrupole-time of flight (Q-Tof) Ultima mass spectrometer equipped with a Z-

spray probe and mass analyser SYNAPT (in Q-Tof mode; Waters, USA), controlled by

MassLynx v4.1 software (Micromass, UK). The instrument was calibrated using cesium

iodide (CsI; 10 mg mL–1 in 70% propan-2-ol) over the same m/z range. For accurate

mass determination of proteins, ~30 L of protein sample (~10 M) was dialysed

extensively against 0.1% (v/v) formic acid as described in Section 3.2.2.2.

3.2.3 Bulk DNA replication assays

Protein subunits of Pol III HE (β2 clamps, αεθ cores, and γ3δδ’ clamp loader

complex), SSB and 5'-flap-primed M13 ssDNA template were kindly provided by Dr.

Slobodan Jergic (University of Wollongong).

The flap-primed ssDNA template was previously constructed by annealing M13

ssDNA to a 30-fold excess of a primer consisting of a 33-nt complementary segment

preceded by a 36-nt non-complementary flap. The amount of proteins used in the assay

was optimised for the novel SYBR Gold staining method used. The standard coupled

strand extension and Pol III SD reaction assay began by preparation on ice of a mixture

of 1 mM ATP, 0.5 mM of each dNTP, 300 nM β2, 925 nM SSB, 150 nM αεθ, 60 nM

γ3δδ’ complex in replication buffer (25 mM Tris.HCl pH 7.6, 10 mM MgCl2, 10 mM DTT

and 130 mM NaCl), in a final volume of 13 L. The mixture was treated for 5 min at

room temperate for the clamp loader complex to be established and then cooled in ice

until the primed DNA template (2.5 nM) was added. The reactions were initiated by

47

quickly transferring the mixtures to a 30C water bath. After 20 min, 10 L samples

were taken from the mixture and immediately quenched by addition of LES buffer [DNA

loading dye, final concentration of 0.04% (w/v) bromophenol blue, 0.04% (w/v) xylene

cyanol FF, 5% (v/v) glycerol), plus 500 mM EDTA and 10% (w/v) of SDS]. The

quenched mixtures were loaded onto a 0.8% agarose gel in 2 x TBE [180 mM Tris-

borate, 4 mM EDTA] running buffer. Products were separated by agarose gel

electrophoresis as described in Section 3.2.1.2 and the gel was run at 55 V for 3 h. The

gel was stained by SYBR Gold nucleic acid gel stain (Invitrogen, USA) and DNA bands

were visualized under 302-nm UV light using a Gel Doc XR+ system (Bio-Rad). The

M13 ssDNA template was loaded in one lane as a reference, as well as a sample of

GeneRuler 1kb DNA Ladder, 250–10,000 bp (Thermo Scientific).

48

4 CONCLUSION

The main aim of this project was to probe the τ subunit and β2 sliding clamp

interaction within the DNA polymerase III holoenzyme. To address this goal,

mutagenesis studies were performed for the τC16 subunit amino acid residues that were

suggested to interact with the clamp (Section 1.4). The τC16R561A+H562A protein

presented the successfully purified 16.372 kDa subunit of the clamp loader complex

containing the mutations of the residues in the desired sites. Functionality of the

τC16R561A+H562A double point mutations was assessed in vitro by a bulk strand

displacement assay (Section 2.1.5). In this study, τC16R561A+H562A was observed to

moderately disturb the process of bacterial DNA replication. This inhibition was

proposed to be a result of a weaker binding to α or β2. However, τc16R561A+H562A

showed a minor difference compared to wild type τC16 and hence, no big conclusions

could be drawn from this assay. Further testing of the τC16 proteins that contain a

mutation to an oppositely charged residue may provide a greater effect on function.

Nevertheless, under these consistent conditions, the other purified τC16 mutants

(τC16D592A, τC16H562A and τC16R561E+H562A) underwent proteolysis in vivo to yield a

final product with a molecular weight of ~15 kDa, as assessed by SDS-PAGE and ESI-

MS.

Due to ambiguous τC16Δ7 proteolysis in vivo, future research should consider this

tendency for degradation and improve the methods used to overproduce and maintain

the purity of these mutated proteins. Upon successful confirmation of the six full-length

τC16 mutants, the function of each variant should be tested and compared in in vitro bulk

replication assays, thus providing conclusive evidence for the polarization effect of each

mutated residue on bacterial DNA replication.

Subsequently, lethality of all τC16 mutants should be tested on E. coli cells. By

cloning the mutant into a pET vector that τC16 is toxic in, transformation efficiency of the

constructed plasmids can be observed. If transformation occurs, it implies that the

mutant is less toxic than wild type τC16, and thus, it shows a weaker binding affinity to α.

Generally, this bulk strand displacement assay did not provide significant

information about the potential τ–β2 interaction, and thus, further examination in the

future will be required to resolve this matter. The most significant studies would involve

49

the expression and purification of the full-length τ mutants and their assembly to the

clamp loader complexes. This should lead to the in vitro reconstitution and purification

of the full CLC, which will considerably assist with the determination of the functional

effect of the τ subunit. Once the full clamp loader containing mutated residues has been

assembled, a bulk replication assay could be used to determine its overall effect in

replication.

However, recent studies have shown a weak interaction, in the absence of DNA,

between α and the τ residues that are prone to interact with the β2 sliding clamp, which

increases the difficulty to interpret the τ–β2 interaction55. If an effect was observed in the

bulk assay, single molecule studies could be performed to determine whether these

functional effects arise from the interaction of τ with α or β2. The β2 sliding clamp is

needed for rapid and highly processive DNA synthesis, and once it is assembled onto

DNA by the clamp loader complex, its interaction with α confers efficient synthesis on

all core polymerases. Therefore, binding of τ to the clamp would be expected to affect

the processivity and rate in a bead experiment, while interacting with α affects the

loading onto DNA, and thus the replication efficiency.

In conclusion, the future proposed experiments should provide a comprehensive

understanding of the individual structures and functions of the DNA Pol III HE subunits

and their role in the essential process of DNA replication. Additionally, a deeper insight

of these mechanisms, specifically the binding within the β2 sliding clamp, could enhance

novel antibacterial drug design targeted at proteins with low-resistance probability.

50

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