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

1 Maturation of Okazaki fragments

Topoisomerases control chromosome topology Catenanes/knots

Topos

Relaxed/disentangled

•Major therapeutic target - chemotherapeutics/antibacterials

•Type II topos transport one DNA through another

2 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

3 Different origin sequences in different organisms

E. Coli (bacteria) OriC

Yeast ARS (Autonomously Replicating Sequences)

Metazoans ????

Initiation in prokaryotes and eukaryotes Bacteria Eukaryotes ORC + other proteins load MCM 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

4 Initiation mechanism in bacteria -- 1

Crystal structure of DnaA:ATP revealed mechanism of origin assembly

1. 2.

1. DnaA monomer (a) forms a polar filament (b).

2. DNA binding sites occur on the outside of the filament (model).

5 Crystal structure of DnaA:ATP revealed mechanism of origin assembly

1. 2.

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).

Initiation mechanism in bacteria -- 1

6 Initiation mechanism in bacteria -- 2

Initiation proteins in E. coli (bacteria)

7 10 ter sites opposite oriC coordinate the end game

Origin Counterclockwise Clockwise fork fork

Tus protein binds Ter sites and inhibits the DnaB helicase only from Clockwise Counterclockwise one direction!!! fork trap fork trap

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

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

DNA Half life (s) Kd (nM) terB

C6 130 (2 min) 1.6

C6 <7 (FAST/permissive) 53

6900 (115 min, SLOW/ 0.4 C6 nonpermissive)

Releasing C6 springs the trap

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

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

DNA Half life (s) Kd (nM) terB

C6 130 (2 min) 1.6

C6 <7 (FAST/permissive) 53 DnaB 5’ 3’ 6900 (115 min, SLOW/ 0.4 C6 nonpermissive)

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.

9 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

10 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!

Summary: What problems do these proteins solve? Function E. coli SV40 (simian virus 40) Helicase DnaB T antigen Primase Primase (DnaG) pol α primase Primer removal pol I’s 5’-3’exo FEN 1 (also RNaseH) Polymerase

Core pol III (α, ε, θ subunits) pol δ, ε Clamp loader γ complex RF-C Sliding clamp β 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

11 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!

Telomere binding proteins (TBPs) 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.

12 Telomerase replicates the ends (telomeres)

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

The RNA templates Telomerase extends DNA synthesis. The the leading strand! proteins include the Synthesis is in the telomerase reverse 5’-->3’ direction. transcriptase TERT.

Telomerase cycles at the telomeres

TERT protein

TER RNA template Telomere ssDNA

13 Telomerase extends a chromosome 3’ overhang

Conserved structures in TER and TERT

Core secondary 148-209 nucleotides structures shared in ciliate and vertebrate telomerase RNAs 1000s of nucleotides (TERs). (Sequences highly variable.)

1300 nucleotides

TERT protein sequences conserved

14 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 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.

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