4.5.Rep.Forks.Ppt [Read-Only]

1. DNA replication is semi-conservative. 2. DNA polymerase enzymes are specialized for different functions. 3. DNA pol I has 3 activities: polymerase, 3’-->5’ exonuclease & 5’-->3’ exonuclease. 4. DNA polymerase structures are conserved. 5. But: Pol can’t start and only synthesizes DNA 5’-->3’! 6. Editing (proofreading) by 3’-->5’ exo reduces errors. 7. High ﬁdelity is due to the race between addition and editing. 8. Mismatches disfavor addition by DNA pol I at 5 successive positions. The error rate is ~1/109.

Replication fork summary

2. Replication fork is organized around an asymmetric, DNA- polymerase III dimer. 3. Both strands made 5’-->3’. 4. “Leading strand” is continuous; “lagging strand” is discontinuous.

1 DNA polymerase can’t replicate a genome!

1. No single stranded template 2. The ss template is unstable 3. No primer 4. No 3’-->5’ polymerase 5. Too slow and distributive

Solution: the replication fork

1. No single-stranded template 2. The ss template is unstable 3. No primer 4. No 3’-->5’ polymerase 5. Too slow and distributive

Schematic drawing of a replication fork

2 DNA polymerase holoenzyme

DNA replication factors were discovered using “temperature sensitive” mutations 1. No single stranded template 2. The ss template is unstable 3. No primer 37 ºC 4. No 3’-->5’ polymerase. 5. Too slow in vitro.

Mutations that inactivate 42 ºC the DNA replication machinery are lethal.

42 ºC, Temperature sensitive Mutant gene (conditional) mutations overexpressed allow isolation of mutations in essential genes.

3 A hexameric replicative helicase unwinds DNA ahead of the replication fork

Helicase assay 1. No single stranded template 2. The ss template is unstable ds DNA 3. No primer 4. No 3’-->5’ polymerase. 5. Too slow in vitro.

Replicative DNA helicase is called DnaB in E. coli.

DnaB couples ATP binding and hydrolysis to DNA strand separation. ss DNA

SSB (or RPA) cooperatively binds ss DNA template

1. No single stranded template 2. The ss template is unstable SSB (single-strand binding protein (bacteria)) or RPA (Replication 3. No primer Protein A (eukaryotes)): 4. No 3’-->5’ polymerase. No ATP used. 5. Too slow in vitro. Filament is substrate for DNA pol.

ss DNA + SSB

ds DNA

4 SSB tetramer structure

1. No single stranded template 2. The ss template is unstable SSB (bacteria) and RPA (eukaryotes) form tetramers. 3. No primer The C-terminus of SSB binds 4. No 3’-->5’ polymerase. replication factors (primase, clamp 5. Too slow in vitro. loader (chi subunit))

C C

N N N N

ss DNA + SSB C C

ds DNA Conservation Positive potential

DNA synthesis is primed by a short RNA segment

1. No single stranded template 2. The ss template is unstable 3. No primer Primase: DNA-dependent RNA 4. No 3’-->5’ polymerase. polymerase 5. Too slow in vitro.

Primase makes about 10-base RNA. The product is a RNA/DNA hybrid. RNA primer has a free 3’OH.

Start preference for CTG on template

Uses ATP, which ends up across from T in the RNA/DNA hybrid.

5 DnaG primase defines a distinct polymerase family (DNA dependent RNA pol)

Model of Ribbon “primosome”: diagram DnaB helicase + DnaG primase

DnaB helicase Map of surface charge

DnaG primase

Primase passes the primed template to DNA polymerase

Leading strand: continuous

Lagging strand: discontinuous

6 DNA pol III “holoenzyme” is asymmetric

1. No single stranded template DNA pol III holoenzyme: 2. The ss template is unstable A molecular machine 3. No primer 4. No 3’-->5’ polymerase. 5. Too slow in vitro.

Synthesizes Synthesizes Leading Lagging Strand Strand

χ binds SSB δ opens clamp (β)

Pol III dimer couples leading and lagging strand synthesis

Leading Lagging strand strand

7 Replication fork

1. No single stranded template 2. The ss template is unstable 3. No primer 4. No 3’-->5’ polymerase 5. Too slow and distributive

Replication fork

1. No single stranded template 2. The ss template is unstable 3. No primer 4. No 3’-->5’ polymerase 5. Too slow and distributive

8 Sliding clamp wraps around DNA

Sliding clamps are structurally conserved

“Palm”

9 Summary of the replication fork

“Palm”

Synthesis of Okazaki fragments by pol III holoenzyme

When pol III reaches the primer of the previous Okazaki fragment, clamp loader removes β2 from the DNA template. As a result, the pol III on the lagging strand falls off the template.

Clamp loader places β2 on the next primer-template.

10 Replication fork 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 -

2. Replication fork is organized around an asymmetric, DNA- polymerase III dimer. 3. Both strands made 5’-->3’. 4. “Leading strand” is continuous; “lagging strand” is discontinuous.

Replication fork summary

1. DNA polymerase can’t replicate a genome. Problem 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 positive Topoisomerase II + supercoils

2. DNA replication is fast and processive

11 Sliding clamp wraps around DNA

γ/RFC clamp loader complex puts the clamp on DNA

6. Sliding clamp can’t get on γ complex -- bacteria 7. Lagging strand contains RNA RFC -- eukaryotes 8. Lagging strand is nicked (Replication Factor C) 9. Helicase introduces + supercoils

12 RFC reaction

1. RFC + clamp + ATP opens clamp 2. Ternary complex + DNA/RNA --> Closed clamp + RFC + ADP + Pi

Schematic drawing of the RFC:PCNA complex on the primer:template

RFC contains 5 similar subunits that spiral around RFC DNA. The RFC helix tracks the DNA or DNA/RNA helix PCNA

DNA:RNA

13 RFC:PCNA crystal structure

RFC

PCNA

DNA:RNA RFC:PCNA crystal structure

SSB opens hairpins, maintains processivity and mediates exchange of factors on the lagging strand

1. No single stranded template SSB (bacteria) and RPA (eukaryotes) 2. The ss template is unstable form tetramers. The C-terminus of SSB binds 3. No primer replication factors (Primase, Clamp 4. No 3’-->5’ polymerase. loader (chi subunit)) 5. Too slow in vitro.

SSB:DNA Primer:template:SSB Clamp loader exchanges binds primase Binds clamp loader with pol III on the clamp Primase - to - pol III switch

14 Synthesis of Okazaki fragments by pol III holoenzyme

DNA polymerase 5’-->3’ exonuclease or RNase H remove RNA primers 6. Sliding clamp can’t get on 7. Lagging strand contains RNA 8. Lagging strand is nicked 9. Helicase introduces + supercoils

DNA polymerase I 5’-->3’ exo creates ss template. Pol works on the PREVIOUS Okazaki fragment!

OR RNaseH cleaves RNA:DNA --> ssDNA + rNMPs primer

15 DNA polymerase 5’-->3’ exonuclease or RNase H remove RNA primers 6. Sliding clamp can’t get on 7. Lagging strand contains RNA 8. Lagging strand is nicked 9. Helicase introduces + supercoils

DNA polymerase I 5’-->3’ exo creates ss template. Pol works on the PREVIOUS Okazaki fragment!

OR RNaseH cleaves RNA:DNA --> ssDNA + rNMPs primer

DNA ligase seals the nicks

1. Adenylylate the enzyme

2. Transfer AMP to the PO4 at the nick

3. Seal nick, releasing AMP

Three steps in the DNA ligase reaction

16 Maturation of Okazaki fragments

All tied up in knots

6. Sliding clamp can’t get on 7. Lagging strand contains RNA 8. Lagging strand is nicked 9. Helicase introduces + supercoils

17 “Topological” problems in DNA can be lethal

(+) supercoils

(-) supercoils •Gene (+) supercoils misexpression

•Chromosome breakage

•Cell death precatenanes

catenanes

Topoisomerases control chromosome topology Catenanes/knots

Topos

Relaxed/disentangled

•Major therapeutic target - chemotherapeutics/antibacterials

•Type II topos transport one DNA through another

18 Topoisomerases cut one strand (I) or two (II)

Topoisomerase I - Cuts ssDNA region (1A (proks)) or nicks DNA (1B (euks))

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

Topoisomerase II is a dimer that makes two staggered cuts

Tyr OH attacks PO4 and forms a covalent intermediate

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

19 Type IIA topoisomerases comprise a homologous superfamily

ATPase DNA Binding/Cleavage

GyrB GyrA

Gyrase Topo II (proks) (euks)

Type IIA topoisomerase mechanism

T-segment

G-segment

1 2 ADP

4 3 • “Two-gate” mechanism • Why is the reaction directional? • What are the distinct conformational states?

20 Summary of the replication fork

“Fingers” “Thumb”

“Palm”

Accessory factors summary

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 positive Topoisomerase II + supercoils

2. DNA replication is fast and processive