5'

3'

3'

5'

5'

5'

DNA Polymerase IIIacts here

DNA Polymerase I extendsone Okazaki fragment and

removes the RNA fromanother.

DNA Ligase then joinsfragments together.

ssDNA bindingprotein (SSB)

Helicase (DnaB)

Primase3'

Gyrase

Spinningat 10,000rpm

A prokaryotic fork is travelling at 50 to 100 kb / minute.

Eukaryotic forks travel at 0.5 - 5 kb / minute.

PrimosomeA Replisome

Pol III has a dimer of the “core subunits”, which contain the polymerizing α subunits.

Core

Pol III*

complex

Fig. 21.17

.

“Clamps” subunit onto DNA, and makes it highly processive.

Donut-shapedDimer.

Fig. 21.15 in Weaver

- Clamp – exists free and as subunit of Pol III holoenzyme

Fig. 21.16

The effect of subunit on the clamp.

Can the clamp can slide off the end of linear DNA?

Based on Fig. 21.13

clamp

Plasmid DNA with nick

Assay 1. Load clamp onto circular plasmid DNA.2. Treat DNA further.3. Separate DNA-bound clamp from free clamp.

Fig. 21.11

Blue – controlRed – treated with the indicated enzyme before chromatography

First peak = protein-DNA complexSecond peak = free protein

Based on Fig. 21.13 f,g

Clamp sliding off the ends of linear DNA can be stopped by DNA binding proteins such as SSB and EBNA.Clamp will slide off SSB-coated DNA if it is part of the holoenzyme that is replicating DNA.

Yellow line- control red line- experimental

SSB can retain clamp, but linearize again after loading SSB, clamp falls off.

Load holoenzyme onto DNA with SSB, if all 4 dNTPs added, clamp falls off (control- only 1 or 2 dNTPs retains clamp)

Pol III core dimer synthesizing leading & lagging strands.

(tau) subunits (2) of Pol III bind to helicase.

Clamp loading

complex of Pol III holoenzyme ( ’, , )

1. Uses ATP to open dimer and position it at 3’ end of primer.

2. “Loaded” clamp then binds Pol III core (and releases from ).

3. Processive DNA synthesis.

- loads subunit dimer onto DNA (at the primer) and Pol core (and unloads it at the end of Okazaki fragment)

Order of events:

Recycling phase

1. Once Okazaki fragment completed, clamp releases from core.

2. binds to 3. unloads clamp from DNA.4. clamp recycles to next primer.

Figure 21.25

Terminating DNA synthesis in prokaryotes.

Fig. 21.26

Each fork stops at the Ter regions, which are 22 bp, 3 copies, and bind the Tus protein.

Decatenation of Daughter DNAs

Fig. 21.27

Decatenation is performed by Topoisomerase IV in E. coli.

Topo IV is a Type II topoisomerase: breaks and rejoins 2 strands of a duplex DNA.

catenane

DNA replication in Eukaryotes

Eukaryotic DNA polymerases (5):

- has primase activity-elongates primers, highly processive, can do

proofreading - DNA repair - DNA repair- replication of Mitochondrial (and/or

Chloroplast DNA in plants)

Eukaryotic DNA polymerases do NOT have 5' to 3' exonuclease activity. A separate enzyme, called FEN-1, is the 5' to 3' exonuclease that removes the RNA primers.

Eukaryotes also have equivalents to the:

Sliding clamp – PCNA (a.k.a. proliferating cell nuclear antigen)

SSB – RP-A

3'5'

DA B C

A' B' D'C'3'5'

DA B C

A' B' D'C'

3'5'

DA B C

A' B' D'C'

Problem for eukaryotes: Replicating the 5’ end of the lagging strand (because chromosomes are linear molecules)

Gap generated by removal of the RNA primer

3'5'

D

D'

A B C

A' B' C'

3'5'

D

D'

A B C

A' B' C'

Euk. chromosomes end with many copies of a special “Telomeric” sequence.

Cells can lose some copies of the telomere w/out losing genes.

(3 copies on this chromosome end)

(Replication of this chromosome would produce 1 that is shorter by 1 telomere)

.

GGG---GGG

GGG

3 ' H O

CCC5 ' P

telomere{GGG---

CCC---

D

D'

A B C

A' B' C'

5'3'

Organism telomere repeatTetrahymena, Paramecium, Oxytricha (allare protozoa)

T2G4

Saccharomyces (yeast) (TG)1-3TG2-3

Arabidopsis (plant) T3AG3

Homo sapiens T2AG3

Telomeres form an unusual secondary structure.

Telomere Sequences

5’ 3’

Dashes are Ts

Enzyme that adds new telomeric repeats to 3’ ends of linear chromosomes.

Diagram of how telomerase works.AAACCCAAAC

3' 5'

GGGTTTGGGTTTGGG

CCCAAACCC||||||||||||||||||

3'5'

Represents the RNA component of telomerase.Protein is not shown.

Represents the end of a chromosome. You'veseen this before.

GGGTTTGGGTTTGGG

CCCAAACCC||||||||||||||||||||||||

3'AAACCCAAAC

3' 5'

GGGTTTGGGTTTGGGTTTG

CCCAAACCC||||||||||||||||||||||||||||

3'AAACCCAAAC

3' 5'

RNA component of telomerase base pairs withend of chromosome as shown.

Telomerase synthesizes new DNA using the RNAcomponent as template.

GGGTTTGGGTTTGGGTTTG

CCCAAACCC||||||||||||||||||||||

3'AAACCCAAAC

3' 5'

GGGTTTGGGTTTGGGTTTGGGTTTG

CCCAAACCC||||||||||||||||||||||||||||

3'AAACCCAAAC

3' 5'

Telomerase moves down and RNA componentbase pairs with end of telomere.

Telomerase synthesizes new DNA using the RNAcomponent as template.

Fig. 21.32 Proteins bind the 3’ SS overhang for protection.

More on the importance of Telomerase

• Apoptosis - Cells are very sensitive to chromosome ends because they are highly recombinogenic. Telomeres don’t trigger apoptosis.

• Aging - There are rapid aging diseases (e.g., Werner’s Syndrome) where telomeres are shorter than

normal.• Cancer - Most somatic cells don’t have telomerase, but

tumor cells do. Over-expression of telomerase in a normal cell, however, won’t turn it into a tumor cell.

• Plants - Transgenic Arabidopsis with the telomerase gene turned off developed normally up to a point, then became sick.

How is a Repl. origin selected?

Priming at the oriC (Bacterial) Origin

GGATCCTGgnTATTAAAAAGAAGATCTnTTTATTTAGAGATCTGTTnTATT Consensussequence GG . . .. A . . C Escherichia G GC . . .. T . . C Salmonella AG . . .. - . . C Enterobacter AG . . .. - . . T Klebsiella CGT A T GA T A C - Erwinia 13 9aGTGATCTCTTATTAGGATCGGnnntnnnnTGTGGATAAgnngGATCCnnnn Consensussequence.. CACTGCCC CAAG GGCT.. CGCCAGGC CCCG TGTA.. ACTCTCTA GTCG ACGA.. GCTTGTCT GTCA GCGGA- TCGTGTTG GTGATTATTCATA

TTtAAGATCAAnnnnnTggnAAGGATCncTAnCTGTGAATGATCGGTGAT Consensussequence. T . CAACC GGA... AT..AA A . TGCGT GGA... AC..G. T . ACGCT AAG... ACA.T. T G CCGTT AAG... GC.TT. A . GAGAA GGCGTT CT..C 9bCCTGGnCCGTATAAGCTGGGATCAnAATGngGGnTTATACACAgCtCAAA Consensussequence ...A G . AG..G . A.T G..T A C GGTAC . A.T ..TT G . AA G G G.T ...T A A . AA G . G.A .A.C TT . TG T . GGA

9bAAncgnACaaCGGTTaTTCTTTGGATAACTACCGGTTGATCCAAGCTTTt Consensussequence .CTGA. AA.A G .. . . . ... .......CC .GTGA. AA.. A .. . . . ... ........C .GCAT. TC.. A .. . . . ... ........T TTCAGG AA.. A A. . . . ... ........T .ACGC. TCG. G .A G G C TTA ACCAGAA.T

9bnAnCAgAGTTATCCACAntnGAnnGcnn-GAT ConsensussequenceTGA G.. GTA. TC.CAC-. EshcerichiaC.C G.T ATG. TC.CAC-. SalmonellaG.G GG. GAA. AGCTGCG. EnterobacterA.G T.. GAA. AA.TAT-. KlebsiellaA.G T.. TTCA CT.CCG-A Erwinia

Three 13-mersGATCTnTTTATTTGATCTnTTnTATTGATCTCTTATTAG

9a-merTGTGGATAA

Three 9b-mersTTATACACATTTGGATAATTATCCACA

Primase (purple) with the first primers (arrows).

Sequence of Events at the Replication Origin

1. Several copies of dnaA bind the four 9-mers; DNA wraps around dnaA forming “Initial Complex”. This requires ATP and a protein, Hu,that is already bound to the DNA.

3. Two copies of dnaB (helicase) bind the 13-mers. This requires dnaC (which does not remain with the Prepriming Complex) and ATP.

4. Primase binds to dnaB (helicase) and the DNA.

2. This triggers opening of the 13-mers (Open complex).

5. dnaB:primase complex moves along the template 5’ > 3’ synthesizing RNA primers for Pol III to extend.

Order of events at OriC