DNA polymerase summary 1 DNA replication is semiconservative

DNA polymerase summary 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 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 1. 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.

DNA polymerase can’t replicate a genome! 1. 2. 3. 4. 5. No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase Too slow and distributive

Solution: the replication fork 1. 2. 3. 4. 5. No single-stranded template The ss template is unstable No primer No 3’-->5’ polymerase Too slow and distributive Schematic drawing of a replication fork

DNA polymerase holoenzyme

DNA replication factors were discovered using “temperature sensitive” mutations 1. 2. 3. 4. 5. No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase. Too slow in vitro. 37 ºC Mutations that inactivate the DNA replication machinery are lethal. 42 ºC Temperature sensitive (conditional) mutations allow isolation of mutations in essential genes. 42 ºC, Mutant gene overexpressed

A hexameric replicative helicase unwinds DNA ahead of the replication fork 1. 2. 3. 4. 5. No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase. Too slow in vitro. Helicase assay ds DNA Replicative DNA helicase is called Dna. B in E. coli. Dna. B couples ATP binding and hydrolysis to DNA strand separation. ss DNA

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

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

DNA synthesis is primed by a short RNA segment 1. 2. 3. 4. 5. No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase. 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. Primase: DNA-dependent RNA polymerase

Dna. G primase defines a distinct polymerase family (DNA dependent RNA pol) Ribbon diagram Model of “primosome”: Dna. B helicase + Dna. G primase Dna. B helicase Map of surface charge Dna. G primase

Primase passes the primed template to DNA polymerase Leading strand: continuous Lagging strand: discontinuous

DNA pol III “holoenzyme” is asymmetric 1. 2. 3. 4. 5. No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase. Too slow in vitro. DNA pol III holoenzyme: A molecular machine Synthesizes Leading Strand Synthesizes Lagging Strand binds SSB opens clamp ( )

Pol III dimer couples leading and lagging strand synthesis Leading strand Lagging strand

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

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

Sliding clamp wraps around DNA N C

Sliding clamps are structurally conserved “Palm”

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 2 from the DNA template. As a result, the pol III on the lagging strand falls off the template. Clamp loader places 2 on the next primer-template.

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 - 1. Sliding clamp can’t get on Clamp loader ( /RFC) + Lagging strand contains RNA Pol I 5’-->3’ exo, RNAse. H Lagging strand is nicked DNA ligase + Helicase introduces positive Topoisomerase II + supercoils

Sliding clamp wraps around DNA N C

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

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 DNA. The RFC helix tracks the DNA or DNA/RNA helix RFC PCNA DNA: RNA

RFC: PCNA crystal structure RFC PCNA RFC: PCNA crystal structure DNA: RNA

SSB opens hairpins, maintains processivity and mediates exchange of factors on the lagging strand 1. 2. 3. 4. 5. No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase. Too slow in vitro. SSB: DNA binds primase SSB (bacteria) and RPA (eukaryotes) form tetramers. The C-terminus of SSB binds replication factors (Primase, Clamp loader (chi subunit)) Primer: template: SSB Binds clamp loader Clamp loader exchanges with pol III on the clamp Primase - to - pol III switch

Synthesis of Okazaki fragments by pol III holoenzyme

DNA polymerase 5’-->3’ exonuclease or RNase H remove RNA primers 6. 7. 8. 9. Sliding clamp can’t get on Lagging strand contains RNA Lagging strand is nicked Helicase introduces + supercoils DNA polymerase I 5’-->3’ exo creates ss template. Pol works on the PREVIOUS Okazaki fragment! OR RNase. H cleaves RNA: DNA --> ss. DNA + r. NMPs primer

DNA polymerase 5’-->3’ exonuclease or RNase H remove RNA primers 6. 7. 8. 9. Sliding clamp can’t get on Lagging strand contains RNA Lagging strand is nicked Helicase introduces + supercoils DNA polymerase I 5’-->3’ exo creates ss template. Pol works on the PREVIOUS Okazaki fragment! OR RNase. H cleaves RNA: DNA --> ss. DNA + r. NMPs primer

DNA ligase seals the nicks 1. Adenylylate the enzyme 2. Transfer AMP to the PO 4 at the nick 3. Seal nick, releasing AMP Three steps in the DNA ligase reaction

Maturation of Okazaki fragments

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

“Topological” problems in DNA can be lethal (+) supercoils (-) supercoils (+) supercoils • Gene misexpression • Chromosome breakage precatenanes • Cell death

Topoisomerases control chromosome topology Catenanes/knots Topos Relaxed/disentangled • Major therapeutic target - chemotherapeutics/antibacterials • Type II topos transport one DNA through another

Topoisomerases cut one strand (I) or two (II) Topoisomerase I - Cuts ss. DNA region (1 A (proks)) or nicks DNA (1 B (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 PO 4 and forms a covalent intermediate Structural changes in the protein open the gap by 20 Å!

Type IIA topoisomerases comprise a homologous superfamily ATPase DNA Binding/Cleavage Gyr. B Gyrase (proks) Gyr. A Topo II (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?

Summary of the replication fork “Fingers” “Palm” “Thumb”

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 - 1. Sliding clamp can’t get on Clamp loader ( /RFC) + Lagging strand contains RNA Pol I 5’-->3’ exo, RNAse. H Lagging strand is nicked DNA ligase + Helicase introduces positive Topoisomerase II + 1. supercoils