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Bacterial DNA replication

Summary: What problems do these proteins solve?

Tyr OH attacksPO4 and forms acovalentintermediate

Structuralchanges in theprotein open thegap by 20 Å!

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DNA ligase IDNA ligaseLigasetopo IItopo IVDecatenationtopo I or topo IIgyraseRemove +sc at fork (swivel)

RF-ASSBssDNA binding

PCNAβ Sliding clampRF-Cγ complex Clamp loaderpol δ, εpol III (α, ε, θ subunits) Core

Polymerase

pol α primaseFEN 1 (also RNaseH)

Primase (DnaG)pol I’s 5’-3’exo

PrimasePrimer removal

T antigenDnaBHelicase

SV40 (simian virus 40)E. coliFunction

… other model systems include bacteriophage T4 and yeast

Summary: What problems do these proteins solve?

The ends of (linear) eukaryotic chromosomescannot be replicated by the replisome.

Not enoughnucleotides forprimase to startlast laggingstrand fragment

Chromosome endsshorten everygeneration!

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Telomere shortening signals trouble!

1. Telomere shortening releasestelomere binding proteins (TBPs)

2. Further shortening affectsexpression of telomere-shortening sensitive genes

3. Further shortening leads toDNA damage and mutations.

Telomere binding proteins (TBPs)

Telomerase replicates the ends (telomeres)

Telomere ssDNA

Telomerase extendsthe leading strand!Synthesis is in the5’-->3’ direction.

Telomerase is aribonucleoprotein(RNP). The enzymecontains RNA andproteins.

The RNA templatesDNA synthesis. Theproteins include thetelomerase reversetranscriptase TERT.

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Telomerase cycles at the telomeres

Telomere ssDNA

TERT protein

TER RNA template

Telomerase extends a chromosome 3’ overhang

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Conserved structures in TER and TERT

Core secondarystructures shared inciliate andvertebratetelomerase RNAs(TERs). (Sequenceshighly variable.)

148-209 nucleotides

1000s of nucleotides

TERT proteinsequences conserved

1300 nucleotides

Starting and stopping summary1. DNA replication is controlled at the initiation step.2. DNA replication starts at specific sites in E. coli and yeast.3. In E. coli, DnaA recognizes OriC and promotes loading of the

DnaB helicase by DnaC (helicase loader)4. DnaA and DnaC reactions are coupled to ATP hydrolysis.5. Bacterial chromosomes are circular, and termination occurs

opposite OriC.6. In E. coli, the helicase inhibitor protein, Tus, binds 10 ter

DNA sites to trap the replisome at the end.7. Eukaryotic chromosomes are linear, and the chromosome ends

cannot be replicated by the replisome.8. Telomerase extends the leading strand at the end.9. Telomerase is a ribonucleoprotein (RNP) with RNA (template)

and reverse-transcriptase subunits.

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DNA methods summary

1. Restriction enzymes cut at specific DNA sites. (N)2. Vectors allow genes to be “cloned” and proteins “expressed”. (N)3. Gel electrophoresis separates DNA on the basis of size.4. DNAs can be synthesized (up to ~100 bases commercially). (N)5. PCR amplifies any target DNA sequence. (N)6. Genes and genomes can be sequenced by chain termination. (N)7. Oligonucleotides can be used to change bases by “site- directed

mutagenesis”. (N)8. “Southern” blotting detects sequences by hybridization.9. Microarrays detect gene expression patterns over the genome.10. Genes can be knocked out (deleted) or replaced in prokaryotes and

eukaryotes. (N)

Restriction enzymes cut DNA at specific sites

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Restriction enzymes cut DNA at specific sites

• 3 types of ends: 5’ overhang, blunt and 3’ overhang• Cognate methyl transferases protect host genome from digestion.

Restriction-modification systems degrade “foreign” DNA.

Average frequency of restriction sites in“random” DNA sequences

The average occurrence of each sequence = 1/4n,where n = the site length and all bases are equally represented

Site size4

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Average frequency 1/256

1/4,096

1/65,536

(1/4 x 1/4 x 1/4 x 1/4)

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Lots of different recognition sites knownCore four bases

Flanking bases

None

A----T

C----G

G----C

T----A

A simple cloning procedure

1. Cut “insert” and “vector”DNA with a restrictionenzyme

2. Mix and join ends withDNA ligase. The endsshould match forefficient ligation.

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Cloning without DNA ligase

1. Prepare open vector and insertwith the same long “sticky” ends

+ Pol I Klenow fragment + dATP

2. Mix and let the ends anneal.

3. Transform the nicked plasmid.The plasmid is repaired in vivo.

1. Prepare an insert flanked bysites for a site-specific DNArecombinase.

2. Mix insert with the closedvector containing therecipient recombination siteand recombinase enzyme.

+

3. (Have lunch.) Transform.

Ligation-independent cloning “Gateway” cloning

E +

No dT in templateT

T

TTAA

TAT

A

“Vectors” allow DNA sequences to be cloned - 1

Phage λ for cloningbig (7-25 kb) DNA

pieces

Ori + selectablemarker + cloningsite (polylinker)

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“Vectors” allow DNA sequences to be cloned - 2

“Reporter” genes:β-gal, GFP . . .

Shuttle vectors:move genes between

organisms

Expression vectors:Make your favorite protein

“Vectors” allow DNA sequences to be cloned -3

Transient transfection: eukaryotes

Stable transduction

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Gel electrophoresis separates DNA on the basis ofsize

Agarose: big fragments (>300 bp)Acrylamide: smaller fragments, higher resolution

Mobility proportional to log MW.

Chemical DNA synthesis

Sequential rounds ofcoupling, oxidationand deprotection ofthe 5’ OH build upthe oligonucleotide.

3’

5’

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Chemical DNA synthesis

Sequential rounds ofcoupling, oxidationand deprotection ofthe 5’ OH build upthe oligonucleotide.

3’

5’

Frontiers in DNA synthesis

Currently: 100-200 nucleotides routine (Assemble 5 kB)10,000 = largest.

Primer set for the human genome (30,000 genes) ~ $104

Goal 1: Make yeast chromosome 3: 300 kB without errors! (Jeff Boeke)

Goal 2: Assemble 16 X 106 w/o errors for ~$1000 (George Church)

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DNA sequencing by partial chain termination

ddNTPs terminatethe chain

DNA sequencing by partial chain termination

Small amount of ddGTP + excessdGTP partially terminates chains at

Cs in the templateddNTPs terminate

the chain

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DNA sequencing by partial chain termination

1. All fragments start at theprimer

2. All fragments ending in aparticular base have adifferent length and adifferent color tag

3. Separating the mixture ofproducts by size reveals thesequence.

Two strategies for genome sequencingHierarchical Shotgun

Sequencing Sequencing

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PCR (Polymerase Chain Reaction): isolate andamplify any DNA sequence

N cycles amplifies the target sequence 2N-fold.

Copies: 1 2 4 8

Site-directed mutagenesis

1. Anneal divergentmutagenic primers.

2. Replicate entire plasmidwith a DNA pol lacking5’-->3’ exonuclease.

3. Select against parentalstrands.

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Gene replacement in mice -- make donor cells1. Insert drug markers into

genome of ES cells2. Select to enrich for

homologous recombinants

Check insertion site bySouthern blotting

Neor confers resistance to G-418.tkHSV confers sensitivity to ganciclovir.

“Southern” blotting detects DNA sequences byhybridization

1. Digest DNA usingrestriction enzyme(s)

2. Run gel

3. Transfer DNA from gelto (nitrocellulose) paper.

4. Denature DNA, hybridizeprobe DNA, and washoff excess probe.

5. Detect the probe on thepaper. E.g. byautoradiography.

“Northern” blotting detects RNA on the gel.

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Microarrays detect expressed genes byhybridization

1. Label cDNAs with red fluorophore in one condition and greenfluorophore in another reference condition.

2. Mix red and green DNA and hybridize to a “microarray”.3. Relative to the reference, Red=enriched, yellow = =, green = depleted.

Each spot has adifferent syntheticoligonucleotidecomplementary to aspecific gene.

Gene replacement in mice -- germline incorporationTransgenic mice express a new

geneWhich mouse

expresses extra copiesof the growth hormone

gene?

1. Inject ES cells into early embryos, 2. Transfer embryos to foster mother,3. Breed chimeric mice and screen for progeny with mutant germ line,4. Screen progeny DNA for mutation, 5. Mate heterozygotes (X+/X-),6. Screen progeny DNA for KO genotype (X-/X-).

Entire process takes a year.

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Gene replacement in plants -- engineered crops

E.g. “Golden rice” synthesizes β-carotene

DNA methods summary

1. Restriction enzymes cut at specific DNA sites. (N)2. Vectors allow genes to be “cloned” and proteins “expressed”. (N)3. Gel electrophoresis separates DNA on the basis of size.4. DNAs can be synthesized (up to ~100 bases commercially). (N)5. PCR amplifies any target DNA sequence. (N)6. Genes and genomes can be sequenced by chain termination. (N)7. Oligonucleotides can be used to change bases by “site- directed

mutagenesis”. (N)8. “Southern” blotting detects sequences by hybridization.9. Microarrays detect gene expression patterns over the genome.10. Genes can be knocked out (deleted) or replaced in prokaryotes and

eukaryotes. (N)