6.start.stop.07.v2.ppt [Read-Only] - Molecular and Cell Biology |

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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 fork Too 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

Transcript of 6.start.stop.07.v2.ppt [Read-Only] - Molecular and Cell Biology |

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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

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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

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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

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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

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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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Initiation mechanism in bacteria -- 2

Initiation proteins in E. coli (bacteria)

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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.

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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

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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)

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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

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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

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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.