REGULATION OF DNA GLYCOSYLATION BY TWO THYMIDINE HYDROXYLASE

ENZYMES IN TRYPANOSOMA BRUCEI

by

SHANDA RAYE BIRKELAND

(Under the Direction of Robert Sabatini)

ABSTRACT

Base J, or β-D-glucosyl-hydroxymethyluracil, is an unusual DNA base modification

present in the genome of bloodstream form Trypanosoma brucei. The presence of J correlates

with epigenetic silencing of telomeric expression sites regulating antigenic variation. Two

proteins, JBP1 and JBP2, are hypothesized to regulate the first step in J biosynthesis via a

conserved N-terminal AlkB-like thymidine hydroxylase (TH) domain. However, the functional

domains and binding requirements for JBP1 and JBP2 stimulated hydroxylation remain to be

tested. Mutagenesis, domain swapping, and heterologous expression experiments suggest a role

of DNA substrate specificity for thymidine hydroxylation. These data show the JBP2 TH motif

is essential for function and JBP1 and JBP2 are TH enzymes with non-interchangeable domains.

Furthermore, JBP2 homologs show decreased levels of J synthesis in different DNA substrates.

These results highlight fundamental differences in thymidine hydroxylation by JBP1 and JBP2 in

J biosynthesis.

INDEX WORDS: Base J, DNA glycosylation, JBP1, JBP2, Trypanosoma, Brucei, Cruzi,

Leishmania, Thymidine hydroxylation, SWI2/SNF2, Kinetoplastids, AlkB, D-glucosyl-hydroxymethyluracil

REGULATED DNA GLYCOSYLATION BY TWO THYMIDINE HYDROXYLASE

ENZYMES IN TRYPANOSOMA BRUCEI

by

SHANDA RAYE BIRKELAND

B.S., Michigan State University, 2003

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment

of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2008

© 2008

Shanda Raye Birkeland

All Rights Reserved

REGULATED DNA GLYCOSYLATION BY TWO THYMIDINE HYDROXYLASE

ENZYMES IN TRYPANOSOMA BRUCEI

by

SHANDA RAYE BIRKELAND

Major Professor: Robert Sabatini

Committee: Stephen Hajduk Lance Wells

Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2008

iv

ACKNOWLEDGEMENTS

I would like to thank my entire family for all of their support through this whole

experience. Many, many thanks to all the people in the lab for their laughter and not taking me

too seriously: Dilrukshi Ekanayake, Robbie Southern, Marion Waltamath, Blake Willis, Josh

Stripling, Laura Cliffe, Kate Sweeney, and all members of the Hajduk laboratory for their helpful

advice. I would like to thank Bob Sabatini for seeing my potential and taking me into his

laboratory. In addition, I would like to thank my advisory committee, Steve Hajduk and Lance

Wells, for their support, encouragement and guidance throughout this process as I have grown as

a person and as a scientist.

v

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS .......................................................................................................iv

LIST OF FIGURES ...................................................................................................................vi

CHAPTER

1 INTRODUCTION.....................................................................................................1

2 MATERIALS AND METHODS ...............................................................................6

3 RESULTS .................................................................................................................9

4 DISCUSSION .........................................................................................................18

REFERENCES .........................................................................................................................21

vi

LIST OF FIGURES

Page

Figure 1.1: Localization of base J in telomeric VSG expression sites...........................................2

Figure 1.2: Formation of base J in a two-step biosynthesis pathway ............................................3

Figure 1.3: Proteins involved in J biosynthesis ............................................................................4

Figure 3.1: Analysis of DNA and protein expression of JBP2 TH mutant cell lines ...................14

Figure 3.2: Analysis of JBP1 and JBP2 domain swapping proteins............................................15

Figure 3.3: Analysis of JBP1-LacI activity in procyclic cells containing LacO repeats ..............16

Figure 3.4: Analysis of TcJ2 and LmJ2 activity in T. brucei ......................................................17

1

CHAPTER 1

INTRODUCTION

African trypanosomes are parasites that cause African sleeping sickness in humans and

the related disease, Nagana, in cattle. Trypanosoma brucei sp. are responsible for causing these

re-emerging diseases in parts of Africa and is 100% fatal if left untreated. The parasite is

transmitted through the tsetse fly where it lives in the midgut and salivary glands. Upon finding

a suitable host, the fly takes a bloodmeal and injects these parasites into the bloodstream of the

host. The trypanosome is able to survive in the mammalian bloodstream by staying one step

ahead of the immune system by a process called antigenic variation. The outside of the parasite

is covered with a single monolayer of a variant surface glycoprotein, or VSG. Over a period of

time, the host immune system develops a response to the parasite by producing antibodies

against the VSG. However, the parasite is able to switch and express a different VSG antigen

allowing the infection to persist. The genome of T. brucei contains a large repertoire of VSG

genes (~1000) to choose from1, 2. Understanding the mechanism and control of antigenic

variation may be helpful in the development of treatments for these diseases.

To be expressed a VSG gene must be located within a specialized telomeric expression

site. While there are ~20 expression sites, only one is active at a time3. Expression sites are

regulated by transcription by RNA polymerase (pol) I, an unusual feature, as pol II typically

transcribes protein encoding genes4. Regulating mono-allelic expression is crucial in order to

maintain infection.

2

The inactive telomeric expression sites contain a high concentration of an unusual DNA

base modification called base J, or β-D-glucosyl-hydroxymethyluracil. The presence of J

correlates with epigenetic silencing of the VSG expression sites (Figure 1.1). This modified base

is developmentally regulated being present only in bloodstream form trypanosomes and not in

the insect stage trypanosome, indicating a very strong linkage of base J and the infection of the

mammalian host.

Figure 1.1. Localization of base J in telomeric VSG expression sites. Schematic diagram showing the presence of base J located in one active (top) and nineteen inactive (bottom) expression sites in T. brucei. Highly repetitive sequences containing base J are labeled throughout the subtelomeric and telomeric regions as indicated.

The presence of J within the ~19 inactive telomeric VSG expression sites, but not within

the single active expression site has suggested that base J is involved in the repression of

telomeric gene expression and/or DNA recombination and thus, regulation of antigenic

variation5. Other epigenetic modifications such as DNA methylation are known to play a role in

regulation of gene expression and suppressing recombination between repetitive elements6.

Whether J plays a similar role in DNA recombination remains to be investigated.

VSG 7 6 2 1 (1)

(19) X

J J J J

50 bp repeats

Telomeric repeats

70 bp repeats

Expression Site Associated Genes

J J J J J J J

J J J J

VSG 7 6 2 1 J J J J J J J J

3

Base J is also found in other kinetoplastids including Crithidia, T. cruzi, Leishmania sp.

and in a closely related protozoa Diplonema7. Unique to kinetoplastids, base J is found in all

telomeric sequences, however it is primarily localized internally in Euglena8.

Base J is proposed to be synthesized in a two-step pathway (Figure 1.2). In the first step,

a thymidine hydroxylase oxidizes specific thymidine residues generating the intermediate,

hydroxymethyluracil (HMU) in DNA followed by a second step, where a glucose moiety is

added to HMU by a glucosyl-transferase to form base J9, 10.

Figure 1.2. Formation of base J in a two-step biosynthesis pathway. Schematic model demonstrating the synthesis pathway of base J. Step one involves the oxidation of thymidine (dT) residues in DNA forming the intermediate HOMedU. Step two involves addition of a glucose moiety by a glucosyl-transferase, forming base J (dJ).

Two proteins, J-binding protein 1 (JBP1) and JBP2 have recently been shown to be

involved in J-biosynthesis in vivo (Figure 1.3). JBP1 and JBP2 share high homology at their N-

terminus (34% identity). However, no clear domain or motif could be identified within this

region until Yu et al. undertook an extensive bioinformatics approach and was able to find a

shared motif with other members of iron and 2-oxoglutarate (Fe/2OG) dioxygenase family of

enzymes11. Proteins within this family have conserved residues involved in iron and 2-

dT HOMedU ß-D-glucosyl-HOMedU (dJ)

Thymidine- hydroxylase ß-glucosyl-

transferase

4

oxoglutarate binding. Given that this class of proteins is able to function as a wide variety of

enzymes including halogenases and dioxygenases, Yu et al. were able to propose a hypothesis

that JBP1 (and JBP2) were the thymidine hydroxylase enzymes that act in the first step of the

synthesis pathway11. Mutagenesis analysis of these key residues was examined in JBP1 and this

led to inactivation of J synthesis without ablating its ability to bind J DNA. This confirms JBP1

as a putative TH enzyme and member of Fe/2OG enzymes. However, JBP2 has not been

examined. In addition, JBP2 contains a SWI2/SNF2 domain necessary for de-novo synthesis of

J, while JBP1 binds specifically to J-DNA in order to function in propagation of J synthesis. We

propose a model in which chromatin remodeling by JBP2 regulates chromosome site-specific de-

novo synthesis of J with further propagation and maintenance of J by JBP1.

Figure 1.3. Proteins involved in J synthesis. Adapted from Borst and Sabatini12. JBP1 and JBP2 functional domains: TH, thymidine hydroxylase motif, JBP, J-binding domain, SWI2/SNF2, ATP-dependent chromatin remodeling domain.

To further elucidate the function of base J, it is necessary to further mechanistically

characterize how JBP1 and JBP2 are involved in the regulation J synthesis. Mutagenesis

analysis of the proposed TH domain was done to characterize JBP2 as a TH enzyme involved in

J synthesis. In order to characterize the mechanism of JBP1 and JBP2 stimulated thymine-

hydroxylation, we are utilizing the LacO/LacI system to tether each to a new region of

chromosome in procyclic cells that normally lack J. The purpose of this study was to analyze the

functional domains in JBP1 and JBP2 to understand how they regulate J synthesis. Furthermore,

5

we have analyzed the specificity of the TH domain of JBP1 and JBP2 by domain swapping

experiments. The identification and characterization of JBP1 and JBP2 as the two TH enzymes

involved in J biosynthesis, outlined in the studies described here, will allow future studies on the

biological function of the modified base in African trypanosomes.

6

CHAPTER 2

MATERIALS AND METHODS

Trypanosome cell culture and transfection. Wild type bloodstream T. brucei strain

427 (BS) and BS JBP1 -/- trypanosomes were cultured and transfected as previously described13.

Wild type procyclic T. brucei strain 29-13 (PC) trypanosomes were cultured and transfected as

previously described14.

Mutagenesis. Mutations were made using the Quikchange II Site-Directed Mutagenesis

kit (Stratagene). Four conserved residues in the putative TH of JBP2 (H391, D393, H441, R455)

were mutated using local alignments corresponding to residues in the JBP1 TH domain11. These

residues were mutated to alanine using an N-terminal green fluorescent protein (GFP)-tagged

JBP2 construct as template. Mutagenesis was carried out using forward and reverse primers of

the targeted region following the manufactures instructions. All constructs were sequenced and

verified by using an ABI Prism 3100 DNA Analyzer (Applied Biosystems). The mutant

constructs were linearized using NotI and XhoI restriction enzymes before transfection into PC

trypanosomes as previously described14.

Western blotting. Protein lysates from 107 cell equivalents were separated on an 8%

SDS-PAGE gel after resuspension in Laemmli buffer. Samples were loaded onto 8% SDS-

PAGE gels and transferred onto nitrocellulose. The blots were then probed with the appropriate

antisera: Anti-LacI (Millipore), anti-La, anti-Histone H3, or anti-GFP (Invitrogen, polyclonal

A6455). Bound antiserum was detected by the appropriate secondary antibodies conjugated to

horseradish peroxidase (HRP) and visualized by enhanced chemiluminescence (ECL).

7

Quantitation of J levels. In order to quantify genomic levels of J, we used an anti-J

immunoblot as previously described7. Briefly, serially diluted genomic DNA was blotted onto

nitrocellulose and incubated with anti-J antisera. Bound J antibody was detected by secondary

antibody conjugated to HRP and visualized by ECL. DNA loading was verified by hybridizing

the membrane with a 32P labeled random primed tubulin probe.

Generation of JBP1 and JBP2 TH domain swapping proteins. In order to generate

JBP1/JBP2 TH domain swapping proteins, we used a PCR stitching method as previously

described15. Briefly, fragments were amplified using chimeric primers to create separate N-

terminal, C-terminal, and TH pieces from JBP1 and JBP2. Subsequent PCR was performed

generating custom-made full-length constructs using standard PCR conditions. Final TH domain

swapping proteins were verified by sequencing using an ABI Prism 3100 DNA Analyzer

(Applied Biosystems). The TH domain swapped proteins were ligated into a GFP-tagged

construct creating N-terminal GFP fusions. Constructs were linearized with NotI and XhoI

before transfection into PC cells and selected under the appropriate drug selection.

Construction of JBP1-LacI fusion constructs. JBP1 was cloned into a GFP-LacI

construct kindly provided by Miguel Navarro. We modified the GFP-LacI fusion construct by

inserting JBP1 in place of GFP16. This construct contained the streptomycin acetyl transferase

(SAT) gene that was used for selection of transfectants, and contained a tetracycline inducible

promoter system. Southern analysis confirmed the proper insertion of the LacO repeats into

genome and western analyis using anti-LacI confirmed the expression of JBP1-LacI fusion

constructs in the transfacted cell lines.

Anti-J Immunoprecipitation. J-DNA fragments were immunoprecipitated and

quantified by dot-blot analysis as previously described7. Briefly, genomic DNA was digested

8

with XhoI and HindIII and incubated with J-antisera in TBSTE buffer containing 10mM Tris pH

8.0, 150 mM NaCl, 0.2% Tween-20, 2 mM EDTA, 0.1 mg tRNA/ml and 1 mg BSA/ml. The

DNA was immunoprecipitated by the addition of protein G agarose beads, and washed 4 times

before spotting onto nitrocellulose along with a fraction (10%) of the input DNA. The

membrane was hybridized with a 32P labeled random primed LacO probe. The LacO probe was

generated by gel purifying LacO repeats from a construct kindly provided by Miguel Navarro16.

Cloning of T. cruzi and L. major JBP2 genes. JBP2 genes were amplified using

genomic DNA from the Y strain of T. cruzi and the Friedlin strain of L. major. After

amplification, gene products were ligated into a GFP-tagged expression construct, creating an N-

terminal fusion tag. After verification of the gene sequences, the linearized constructs (TcJ2:

NotI and XhoI, LmJ2: ApaI and XhoI) were transfected into PC cells and selected under the

appropriate drug selection.

9

CHAPTER 3

RESULTS

Mutation of the proposed TH domain of JBP2 inhibits J synthesis. In order to test

whether JBP2 is a thymidine hydroxylase enzyme, we mutagenized the predicted catalytic

residues involved in iron and 2-oxoglutarate binding (Fe/2OG). To do this, the four conserved

residues in the putative TH of JBP2 (H391, D393, H441, R455) were mutated to alanine in a

GFP-tagged expression JBP2 construct. We then examined the ability of JBP2 mutants to

stimulate de novo J synthesis when expressed in the PC cells, which normally lack J. Based on

previous analysis of other members of the Fe/2OG family, and JBP1, we expect these mutations

will inhibit thymidine hydroxylation, and thus J synthesis. As a positive control, we mutated a

conserved valine residue (V459A) outside of the catalytic motif. WT JBP2 was used as a

comparison for functional enzyme activity. After selecting positive transfectants, DNA was

isolated, spotted onto nitrocellulose, and probed with an antibody against base J. The blot was

then stripped and probed with tubulin to control for DNA loading. The J-DNA dot blot analysis

indicates mutations H391A, H441A, and R455A ablate de novo J synthesis of JBP2 (Figure

3.1A). However, the D393A mutant stimulates nearly identical levels of J in the DNA in PC

cells as WT JBP2. Western blot analysis using an antibody to detect TbJBP2 shows equal

expression of the mutant JBP2 proteins in the cell lines analyzed (Figure 3.1B). Antisera against

the RNA binding protein La, was used as a loading control. With the exception of the D393A

mutant currently undergoing further analysis, we conclude that these mutants support the overall

10

conclusion that JBP2 represents a thymidine hydroxylase enzyme and is a member of the

Fe/2OG dioxygenase family.

JBP1 and JBP2 TH domain swapping analysis. Now that we have evidence that both

JBP1 and JBP2 are thymidine hydroxylases the question arises is why two TH are needed for J

biosynthesis. In order to address this concept we wanted to examine the specificity of the TH

domain. One approach was to ask whether the TH domains in JBP1 and JBP2 were

interchangeable. JBP1 and JBP2 contain a TH domain having 35% identity at the conserved N-

terminus region spanning ~320 amino acids (aa). Without any structural data available for

JBP1/JBP2 and difficulty in modeling the TH domain due to the low homology with other

Fe/2OG enzymes, we decided to swap the 320 aa homologous region between these two

proteins. Constructs were generated using a PCR stitching method and were ligated into a GFP

containing construct. Figure 3.2A shows a schematic drawing of the newly generated constructs

in relation to the essential residues thought to be involved in iron and 2-oxoglutarate binding and

catalysis. We can address the activity of the TH domain swapped proteins by looking at the

ability of J1-J2TH-J1 to rescue a BS J1 -/- by propagating J levels and J2-J1TH-J2 to stimulate

de novo synthesis upon expression in PC cells. WT JBP1 and JBP2 GFP-fused constructs were

used as positive controls. After selecting positive transfectants, DNA and protein was isolated

for further analysis. Figure 3.2B shows protein levels of JBP1, J1-J2TH-J1, JBP2, J2-J1TH-J2

expressing cell lines using an antibody against GFP. Expression of the J1-J2TH-J1 cell line

shows a lower protein expression level when compared with full length JBP1. Comparison of

protein levels in JBP2 and J2-J1TH-J2 expressing cell lines show no difference. Figure 3.2C

shows DNA analysis of the of TH domain swapping proteins. The BS form J1 -/- cells show a

16-fold decrease in the level of J, which can be rescued to WT levels upon expression of WT

11

JBP1. Therefore, the addition of JBP1 is able to propagate J synthesis and rescue the JBP1 KO

cell line. As mentioned earlier, JBP2 is able to stimulate J synthesis when expressed in PC cells.

However, J2-J1TH-J2 was unable to initiate de novo synthesis of J. Although both TH domain-

swapping proteins show protein expression, neither show the ability to compensate and function

like their WT counterparts. The inability to swap TH domains between JBP1/JBP2 suggests a

difference in TH specificity, common in other Fe/2OG enzymes17, and may explain a reason for

having two TH enzymes involved in regulating the first step in J synthesis.

Analysis of TH specificity of JBP1 via LacI/LacO interaction. JBP1 has the ability to

bind specifically to double-stranded J-DNA18-20, and is involved in propagation of base J within

specialized telomeric expression sites21. Yu et al. hypothesized that JBP1, once bound to J-

DNA, catalyzes thymidine hydroxylation via its N-terminal domain11. However, it remains to be

determined if binding of J-DNA is required for thymidine hydroxylation by JBP1. We decided

to address this by fusing a known DNA binding domain to JBP1 and examine the requirement

for DNA binding and J biosynthesis by JBP1 in the absence of J-DNA. To do this, we expressed

a C-terminal JBP1-LacI fusion protein in PC cells that contain large arrays of the LacO repeats

inserted into the genome. As a negative control, a mutation within the TH motif was made in

JBP1-LacI. After selecting for positive transfectants, DNA and protein was isolated for further

analysis. Using an antibody against LacI, Figure 3.3A shows a western blot of cell lines

expressing JBP1-LacI (WT) and JBP1-LacI H213A (TH mut). After a 2-day tetracycline

induction, both samples show relatively equal expression. The blot was stripped and probed with

anti-La as a loading control. Figure 3.3B shows total DNA analysis of cell lines expressing

JBP1-LacI (WT) and JBP1-LacI (TH mut). JBP1-LacI (WT) shows a modest level of J whereas

the TH mutant does not make any J, as expected. In order to look at specific localization of J,

12

DNA samples were digested to cut out the LacO repeats followed by immunoprecipitation with

anti-J. Samples were then spotted onto nitrocellulose, using LacO repeats as a probe. IP results

show the presence of J in LacO repeats when JBP1-LacI (WT) is expressed, where as JBP1-LacI

(TH mut) served as a negative control.

Heterologous expression of T. cruzi and L. major JBP2 in T. brucei. JBP2 is involved

in de novo synthesis where it specifically localizes J in repeats throughout telomeric expression

sites in T. brucei14. Other J containing kinetoplastids do not have telomeric ES or undergo

antigenic variation, but still have J located in telomeric repeats. L. major and T. cruzi have 98%

and 75% of the total J located within the telomeres7, while the remaining 25% of J in T. cruzi is

located in the subtelomeric regions containing other repetitive regions and genes involved in

pathogenesis22. Interestingly, J DNA in Euglena is primarily located internally in the genome8.

Given the difference in localization of base J in these organisms, it is not known how JBP2 is

able to specify localization of base J the chromatin. In order to address this issue, we decided to

test other JBP2 homologs for their ability to interact with a different substrate. T. brucei, T. cruzi,

and L. major JBP2 genes share high homology. When compared with TbJBP2, TcJBP2 is more

closely related (56% identity) than LmJBP2 (43% identity). Figure 3.4A shows a schematic

representation of the three JBP2 genes used in this study. TcJBP2 and LmJBP2 were amplified

from genomic DNA. Confirmation of the gene sequence was performed before the gene

products were cloned into a GFP-tagged expression construct. Each of the newly generated

constructs were transfected into PC cells. We can monitor the function of TcJBP2 and LmJBP2

by looking at de novo J synthsis levels in PC cells, using TbJBP2 as a positive control. After

selecting for positive tranfectants, DNA and protein was isolated for further analysis. Figure

3.4B shows protein expression levels of the indicated cell lines using a GFP antibody. TbJBP2

13

and TcJBP2 show relatively equal protein levels, whereas LmJBP2 is expressed at a higher level.

Anti-La served as a loading control. Figure 3.4C shows DNA analysis from PC cell lines

expressing TbJBP2, TcJBP2, and LmJBP2; the right panel shows an anti-J dot blot examining

the levels of de novo J synthesis, and the left panel shows the same blot stipped and probed with

tubulin as a loading control. When expressed in PC cells, TcJBP2 makes 8-fold less J when

compared with TbJBP2 while LmJBP2 appears to be inactive. These data suggest a role of JBP2

in DNA or chromatin substrate specificity that may be species specific.

14

Figure 3.1. Analysis of DNA and protein expression of JBP2 TH mutant cell lines. (A) The left panel shows an anti-J dot blot of the indicated DNA samples serially diluted and spotted onto nitrocellulose. A specific antibody against J was then used to detect J levels in these DNA samples. DNA isolated from BS and PC, bloodstream and procyclic form trypanosomes, were used as positive and negative controls. The right panel shows the same blot stripped and probed with tubulin as a loading control. (B) Western blot analysis of the indicated cell lines. To detect expression of the TbJBP2 TH mutant proteins, the blot was probed with a peptide antibody reactive against TbJBP2. The blot was then stripped and probed with anti-La to serve as a loading control.

15

Figure 3.2. Analysis of JBP1 and JBP2 TH domain swapping proteins. (A) Schematic representation of the genes constructed in this study. JBP1 (yellow) and JBP2 (green) can be separated into three individual pieces: N-terminus, homologous TH region, and C-terminus. Solid lines within the TH region indicate catalytic residues. Amino acid numbers are indicated. (B) Western blot analysis of WT and TH domain swapping proteins using an anti-GFP antibody. Anti-Histone H3 was used as a loading control. (C) Anti-J dot blot analysis of WT and TH domain swapping cell lines.

16

Figure 3.3. Analysis of JBP1-LacI activity in procyclic cells containing LacO repeats. (A) Western blot analysis showing expression of JBP1-LacI fusion constructs (WT and TH mut) using an antibody against LacI. (B) Anti-J dot blot showing J levels in cells expressing WT and TH mut JBP1-LacI constructs. (C) Anti-J immunoprecipitation showing localization of J within LacO repeats after expression of WT and TH mut JBP1-LacI fusion proteins.

17

Figure 3.4. Analysis of TcJ2 and LmJ2 activity in T. brucei. (A) Schematic representation of each of the JBP2 genes from T. brucei (Tb), T. cruzi (Tc), and L. major (Lm). (B) Western blot analysis of TbJ2, TcJ2, and LmJ2 cell lines using an anti-GFP antibody. Anti-La was used as a loading control. (C) Right panel: Anti-J dot blot analysis of TbJ2, TcJ2, an LmJ2 expressing cells. Left panel: Blot was stripped and probed with tubulin to serve as a loading control.

18

CHAPTER 4

DISCUSSION

JBP2 represents a TH enzyme involved in J synthesis. Mutagenesis of residues

(H391, H441. R455) hypothesized to be involved in catalysis inhibits activity of JBP2 to

stimulate de novo J synthesis when expressed in PC cells, supporting the results that JBP2 is a

thymidine hydroxylase. Unexpectedly, mutation of residue D393A shows de novo synthesis at

levels comparable to those of WT JBP2. In order to explain this, another residue or ion may play

a role in filling the vacant iron coordination site allowing full activity. Blasiak et al. were able to

solve a structure of an aspartic acid mutant SyrB2, a halogenase enzyme, in which a chlorine ion

was now involved in iron coordination23. Hewitson et al. recently published data indicating that

a mutant FIH (D201A), an asparaginyl hydroxylase, can still bind iron using only two histidine

residues24. These results indicate variation of the catalytic residues thought to be necessary for

iron binding in this large family of Fe/2OG enzymes. In addition, our lab has generated a

bloodstream JBP1/JBP2 -/- cell line which lacks base J altogether, further confirmation that JBP1

and JBP2 are the thymidine hydroxylase enzymes involved in the first step in J biosynthesis (L.

Cliffe et al., unpublished results). The studies presented here strongly support the hypothesis

that JBP2 is a thymidine hydroxylase enzyme, however, these results are indirect and definitive

proof that JBP1/JBP2 are TH enzymes will require development of an in vitro system.

JBP1 and JBP2 domains are not interchangeable. J1-J2TH-J1 is unable to propagate J

in a BS J1 -/- cell line, and J2-J1TH-J2 is incapable of stimulating J synthesis in PC cells. Due to

the size of the domain swap (~320 aa) and lack of structural information used in generating these

19

new fusions, we cannot rule out incorrect or incompatible protein folding leading to inactivity.

In the case of J1-J2TH-J1, the inability to propagate J may be due to low protein expression. It is

also possible that there is a mechanistic linkage between TH activity at the N-terminus, to the C-

terminus: J-DNA binding domain in JBP1 and chromatin interaction via SWI2/SNF2 domain in

JBP2. Attempts to verify binding of JBP1/JBP2 by performing chromatin association

experiments were tried but were unsuccessful. This information will rule out whether inactivity

is due to a failure to bind. The inability to swap TH domains between JBP1/JBP2 suggests a

difference in TH specificity at the DNA substrate level, common in other Fe/2OG enzymes17,

and may explain a reason for having two TH enzymes involved in regulating the first step in J

synthesis.

JBP1 can bypass J-DNA binding and stimulate J synthesis. Fusion of a LacI binding

domain onto JBP1, stimulated J synthesis specifically in LacO repeats when expressed in PC

cells. This suggests binding to J-DNA is not a requirement for JBP1 to propagate J. The amount

of J made by JBP1-LacI in the LacO repeats was not quantified, but the amount of J remains to

be determined, particularly in cells with different size LacO repeats. Further experiments using

JBP2 fused with LacI are

Activity of JBP2 homologs affected by genomic DNA substrate. Expression of TcJ2

in T. brucei shows decreased levels of de novo J synthesis when expressed in PC cells. To

explain these results, optimal chromatin binding by TcJ2 may not be achieved in the context of

different DNA/chromatin substrate. However, crude chromatin association experiments were

unsuccessful and therefore we were not able to look at levels of chromatin association by TcJ2

when expressed in T. brucei. Other methods for chromatin interaction can to be carried out, such

as CHIP (chromatin immunoprecipitation). In addition, localization of J in TcJ2/PC remains to

20

be analyzed before any further conclusions regarding low levels of de novo synthesis can be

drawn from chromatin interaction.

LmJ2 is unable to synthesize J when expressed in T. brucei PC cells. Crude chromatin

association experiments were unsuccessful, and thus we were unable to rule out the inability to

initiate de novo synthesis was due to the inability to associate with PC chromatin. Until further

chromatin association/binding experiments are successful, it remains unclear why LmJ2 does not

synthesize J in PC cells. It should be noted that expression of Leishmania genes in T. brucei is

feasible; Leishmania JBP1 expression is able to fully rescue J levels in T. brucei J1 -/-11. These

results bring up another question regarding what role the SWI2/SNF2 domain plays in chromatin

interaction.

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REFERENCES

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