Accessory factors summary 1.DNA polymerase can’t replicate a genome. SolutionATP? No single...

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

(/RFC) +Lagging strand contains RNAPol 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

Starting and stopping summary

1. 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

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

PCNA:pol

MCM (helicase) + RPA (ssbp)

PCNA:pol (clamp loader)

Primase + DNA pol

PCNA:pol DNA ligase

Crystal structure of DnaA:ATP revealed mechanism of 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.

Crystal structure of DnaA:ATP revealed mechanism of origin assembly

1. The arrangement of DNA binding sites introduces positive supercoils by wrapping DNA on the outside.

Compensating negative supercoils melt the replication bubble at the end.

2. Clamp deposition recruits Had, which promotes ATP hydrolysis and progressive disassembly of the DnaA filament (hypothesis).

1. 2.

Initiation mechanism in bacteria -- 1

Initiation mechanism in bacteria -- 2

Initiation proteins in E. coli (bacteria)

10 ter sites opposite oriC coordinate the end game

The ter/tus system is not essential in E. coli.

Tus protein binds Ter sites and inhibits the DnaB helicase

OriginCounterclockwise

forkClockwisefork

Clockwisefork trap

Counterclockwisefork trap

Unwinding ter from the “nonpermissive” direction springs a “molecular mousetrap”

Releasing C6 springs the trap

DNA Half life (s) Kd (nM)

130 (2 min) 1.6

53 (<1 min, FAST/ 53

permissive)

6900 (115 min, SLOW/ 0.4

nonpermissive)

terB

C6

C6

C6

Mulcair et al. (2006) Cell 125, 1309-1319.

Unwinding ter from the “nonpermissive” direction springs a “molecular mousetrap”

Releasing C6 springs the trap

DNA Half life (s) Kd (nM)

130 (2 min) 1.6

53 (<1 min, FAST/ 53

permissive)

6900 (115 min, SLOW/ 0.4

nonpermissive)

terB

C6

C6

C6

5’3’

Mulcair et al. (2006) Cell 125, 1309-1319.

Unwinding ter from the “nonpermissive” direction springs a “molecular mousetrap”

Releasing C6 springs the trap Mulcair et al. (2006) Cell 125, 1309-1319.

Unwinding ter from the nonpermissive direction springs a “molecular mousetrap”

Releasing C6 springs the trap Mulcair et al. (2006) Cell 125, 1309-1319.

Topoisomerase II unlinks the replicated chromosomes

Topoisomerase II - Cuts DNA and passes one duplex through the other.

Class II topoisomerases include:Topo IV and DNA gyrase

Summary: What problems do these proteins solve?

Tyr OH attacks PO4 and forms a covalent intermediate

Structural changes in the protein open the gap by 20 Å!

Function E. coli SV40 (simian virus 40)

Helicase DnaB T antigen

Primase

Primer removal

Primase (DnaG)

pol I’s 5’-3’exo

pol primase

FEN 1 (also RNaseH)

Polymerase

Core pol III (, , subunits) pol ,

Clamp loader complex RF-C

Sliding clamp PCNA

ssDNA binding SSB RF-A

Remove +sc at fork (swivel) gyrase topo I or topo II

Decatenation topo IV topo II

Ligase DNA ligase DNA ligase I

… other model systems include bacteriophage T4 and yeast

Summary: What problems do these proteins solve?

The ends of (linear) eukaryotic chromosomes cannot be replicated by the replisome.

Not enough nucleotides for primase to start last lagging strand fragment

Chromosome ends shorten every generation!

Telomere shortening signals trouble!

1. Telomere shortening releases telomere binding proteins (TBPs)

2. Further shortening affects expression of telomere-shortening sensitive genes

3. Further shortening leads to DNA damage and mutations.

Telomere binding proteins (TBPs)

Telomerase replicates the ends (telomeres)

Telomere ssDNA

Telomerase extends the leading strand!Synthesis is in the 5’-->3’ direction.

Telomerase is a ribonucleoprotein (RNP). The enzyme contains RNA and proteins.

The RNA templates DNA synthesis. The proteins include the telomerase reverse transcriptase TERT.

Telomerase cycles at the telomeres

Telomere ssDNA

TERT protein

TER RNA template

Telomerase extends a chromosome 3’ overhang

Conserved structures in TER and TERT

Core secondary structures shared in ciliate and vertebrate telomerase RNAs (TERs). (Sequences highly variable.)

148-209 nucleotides

1000s of nucleotides

TERT protein sequences conserved

1300 nucleotides

Starting and stopping summary

1. 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.