a replisome. pol iii has a dimer of the “core subunits”, which contain the polymerizing α...
TRANSCRIPT
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
Spinning
at 10,000
rpm
A prokaryotic fork is travelling at 50 to 100 kb / minute.
Eukaryotic forks travel at 0.5 - 5 kb / minute.
Primosome
A 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 repeat
Tetrahymena, 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. 21.32Proteins 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-mers
GATCTnTTTATTTGATCTnTTnTATTGATCTCTTATTAG
9a-mer
TGTGGATAA
Three 9b-mers
TTATACACATTTGGATAATTATCCACA
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