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Transcript of 6.start.stop.07.v2.ppt [Read-Only] - Molecular and Cell Biology |
1
Accessory factors summary
1. DNA polymerase can’t replicate a genome.Solution ATP?
No single stranded template Helicase +The ss template is unstable SSB (RPA (euks)) -No primer Primase (+)No 3’-->5’ polymerase Replication forkToo slow and distributive SSB and sliding clamp -Sliding clamp can’t get on Clamp loader (γ/RFC) +Lagging strand contains RNA Pol I 5’-->3’ exo, RNAseH -Lagging strand is nicked DNA ligase +Helicase introduces + supercoils Topoisomerase II +
and products tangled
2. DNA replication is fast and processive
DNA polymerase holoenzyme
2
Maturation of Okazaki fragments
Topoisomerases control chromosome topologyCatenanes/knots
Relaxed/disentangled
•Major therapeutic target - chemotherapeutics/antibacterials
•Type II topos transport one DNA through another
Topos
3
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 7 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.
Isolating DNA sequences that mediate initiation
4
Different origin sequences in different organisms
E. Coli (bacteria)OriC
YeastARS(Autonomously Replicating Sequences)
Metazoans ????
Initiation in prokaryotes and eukaryotesBacteria Eukaryotes
ORC + other proteins loadMCM hexameric helicases
MCM (helicase) + RPA (ssbp)
Primase + DNA pol α
PCNA:pol δ + RFC
MCM (helicase) + RPA (ssbp)
PCNA:pol δ + RFC (clamp loader)
Primase + DNA pol α
PCNA:pol δ + DNA ligase
5
Initiation mechanism in bacteria -- 1
Crystal structure of DnaA:ATP revealed mechanismof origin assembly
1. DnaA monomer (a) forms a polar filament (b).
2. DNA binding sites occur on the outside of the filament (model).
1. 2.
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Crystal structure of DnaA:ATP revealed mechanismof origin assembly
1. The arrangement of DNA binding sites introduces positive supercoils bywrapping DNA on the outside.
Compensating negative supercoils melt the replication bubble at the end.
2. Clamp deposition recruits Had, which promotes ATP hydrolysis andprogressive disassembly of the DnaA filament (hypothesis).
1. 2.
Initiation mechanism in bacteria -- 1
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10 ter sites opposite oriC coordinate the end game
The ter/tus system is not essential in E. coli.
Tus protein bindsTer sites andinhibits the DnaBhelicase only fromone direction!!!
OriginCounterclockwise
forkClockwisefork
Clockwisefork trap
Counterclockwisefork trap
Unwinding ter from the “nonpermissive” directionsprings a “molecular mousetrap”
Releasing C6 springs the trap
DNA Half life (s) Kd (nM)
130 (2 min) 1.6
<7 (FAST/permissive) 53
6900 (115 min, SLOW/ 0.4 nonpermissive)
terB
C6
C6
C6
Mulcair et al. (2006) Cell 125, 1309-1319.
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Unwinding ter from the “nonpermissive” directionsprings a “molecular mousetrap”
Releasing C6 springs the trap
DNA Half life (s) Kd (nM)
130 (2 min) 1.6
<7 (FAST/permissive) 53
6900 (115 min, SLOW/ 0.4 nonpermissive)
terB
C6
C6
C6
5’3’
Mulcair et al. (2006) Cell 125, 1309-1319.
DnaB
Unwinding ter from the “nonpermissive” directionsprings a “molecular mousetrap”
Releasing C6 springs the trap Mulcair et al. (2006) Cell 125, 1309-1319.
10
Unwinding ter from the nonpermissive directionsprings a “molecular mousetrap”
Releasing C6 springs the trap Mulcair et al. (2006) Cell 125, 1309-1319.
Topoisomerase II unlinks the replicatedchromosomes
Topoisomerase II - Cuts DNAand passes one duplex throughthe other.
Class II topoisomerases include:Topo IV and DNA gyrase
11
Summary: What problems do these proteins solve?
Tyr OH attacksPO4 and forms acovalentintermediate
Structuralchanges in theprotein open thegap by 20 Å!
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?
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The ends of (linear) eukaryotic chromosomescannot be replicated by the replisome.
Not enoughnucleotides forprimase to startlast laggingstrand fragment
Chromosome endsshorten everygeneration!
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)
13
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.
Telomerase cycles at the telomeres
Telomere ssDNA
TERT protein
TER RNA template
14
Telomerase extends a chromosome 3’ overhang
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
15
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.