molecular biology of dna replication and recombination · molecular biology of dna replication and...
TRANSCRIPT
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