Chapter 6

Molecular biology of DNA Replication and Replication and Recombination

OverviewIntroductionSemiconservative replication of double-pstranded DNAModes of replicationUnwinding, stabilization and stress reliefInitiation by a primosome complexChain elongation and proofreadingDiscontinuous replication of the lagging strandp gg gTerminator sequencing of DNAMolecular mechanisms of recombination

“It didn’t escape ourpattention that …”

Problems of initiation, ,elongation, incorporation errors

Initiation:Replicons & origins of replicationReplicons & origins of replicationRNA primers

El tiElongation:5’ to 3’ onlyLeading versus lagging strand

Errors or incorporationErrors or incorporationProofreading

OverviewIntroductionSemiconservative replication of double-pstranded DNAModes of replicationUnwinding, stabilization and stress reliefInitiation by a primosome complexChain elongation and proofreadingDiscontinuous replication of the lagging strandp gg gTerminator sequencing of DNAMolecular mechanisms of recombination

Meselson-Stahl experiment

Equilibrium density gradient q y gcentrifugation

Semi-conservative replication pof DNA in chromosomes

Semi-conservative replication pof DNA in chromosomes Overview

IntroductionSemiconservative replication of double-pstranded DNAModes of replicationUnwinding, stabilization and stress reliefInitiation by a primosome complexChain elongation and proofreadingDiscontinuous replication of the lagging strandp gg gTerminator sequencing of DNAMolecular mechanisms of recombination

Theta replication of circular pchromosomes

Theta replication of circular pchromosomes

Theta replication of circular pchromosomes Rolling circle replication

Rolling circle replicationMultiple origins and bidirectional p greplication in eukaryotes

Multiple origins and bidirectional p greplication in eukaryotes

Replication speed and duration p pin pro- and eucaryotes

Eukaryotes:10-100 nt/sec => days / chr (10^7) 10-100 nt/sec => days / chr. (10 7) => multiple origins of replication

> 5 10 h=> 5-10 hrs

Prokaryotes:1,500 nt / sec => 30 min / genome

20 min. generation inertval !g

OverviewIntroductionSemiconservative replication of double-pstranded DNAModes of replicationUnwinding, stabilization and stress reliefInitiation by a primosome complexChain elongation and proofreadingDiscontinuous replication of the lagging strandp gg gTerminator sequencing of DNAMolecular mechanisms of recombination

Unwinding, stabilization and g,stress relief

Unwinding, stabilization and g,stress relief Overview

IntroductionSemiconservative replication of double-pstranded DNAModes of replicationUnwinding, stabilization and stress reliefInitiation by a primosome complexChain elongation and proofreadingDiscontinuous replication of the lagging strandp gg gTerminator sequencing of DNAMolecular mechanisms of recombination

Initiation by a primosome y pcomplex

RNA primerProcaryotes: Procaryotes:

primase (dnaG)2-5 RNA residues

Eucaryotes: yprimosome (polymerase alpha + 15-20 proteins)p )12 RNA residues + > 20 DNA residues

Initiation by a primosome y pcomplex

OverviewIntroductionSemiconservative replication of double-pstranded DNAModes of replicationUnwinding, stabilization and stress reliefInitiation by a primosome complexChain elongation and proofreadingDiscontinuous replication of the lagging strandp gg gTerminator sequencing of DNAMolecular mechanisms of recombination

Chain elongation and gproofreading

ElongationProcaryotes: polymerase III Procaryotes: polymerase III holoenzyme (2 x DNA polymerase III + > 7 p oteins)+ > 7 proteins)Eucaryotes: polymerase deltaReplication errors: rate and causeProofreading exonuclease 3’ to 5’ Proofreading exonuclease 3 to 5 activity

5’ to 3’ chain elongation Proofreading

Proofreading precludes 3’ to 5’ g pelongation Overview

IntroductionSemiconservative replication of double-pstranded DNAModes of replicationUnwinding, stabilization and stress reliefInitiation by a primosome complexChain elongation and proofreadingDiscontinuous replication of the lagging strandp gg gTerminator sequencing of DNAMolecular mechanisms of recombination

Discontinuous replication of the plagging strand

Leading vs lagging strand, Okazaki fragmentsfragmentsJoining of the precursor fragments

Procaryotes:Procaryotes:Pol I (5’ to 3’ exonuclease activity)+ ligaseg

EucaryotesReplication Protein APol deltaLigase

Leading vs lagging strand, g gg g ,Okazaki fragments

Leading vs lagging strand, g gg g ,Okazaki fragments

DNA joining of precursor j g pfragments - procaryotes

5’ to 3’ exonuclease activity of polII

Removes RNA primerGenerates 5’ P end (vs 5’ PPP end of Generates 5’ P-end (vs 5’ PPP-end of primer)

li+ ligase

DNA joining of precursor j g pfragments - eucaryotes Overview

IntroductionSemiconservative replication of double-pstranded DNAModes of replicationUnwinding, stabilization and stress reliefInitiation by a primosome complexChain elongation and proofreadingDiscontinuous replication of the lagging strandp gg gTerminator sequencing of DNAMolecular mechanisms of recombination

Sequencing of DNAq gDideoxyterminators

Sequencing of DNAq gSanger sequencing

Sequencing of DNAq gSanger sequencing

Massive Parallel Sequencingq gReversible terminators

Massive Parallel Sequencingq gPyrosequencing Overview

IntroductionSemiconservative replication of double-pstranded DNAModes of replicationUnwinding, stabilization and stress reliefInitiation by a primosome complexChain elongation and proofreadingDiscontinuous replication of the lagging strandp gg gTerminator sequencing of DNAMolecular mechanisms of recombination

Recombination: double-strand break and repair model

Recombination: double-strand break and repair model

Recombination: Mismatch repair and gene conversion