1. Lecture 1: Fidelity/Specificity: bioregulation through substrate control of molecular choice Use of biochemistry (assays) and genetics (phenotypes) to define function Lecture 2: Breaking down complex processes into intermediates and subreactions In vivo and in vitro analysis of the players, intermediates, and activities Defining activity dependencies to understand their order and timing DNA Polymerase The Replication Fork and Replisome Breaking down complex processes into intermediates and subreactions

2. DNA Polymerase III Understanding Molecular Mechanisms 5’ 3’ 5’ 3’

3. Understanding Molecular Mechanisms SP S = substrate P = product I I = intermediate A1A1 A2A2 A = activity SP I1I1 I2I2 I3I3 I 4………….. I n A1A1 A2A2 A3A3 A4A4 A n+1 How to structurally characterize intermediates? How to detect and identify intermediates? How to identify the proteins/nucleic acids responsible for the activities?

4. Detecting Intermediates SP I1I1 I2I2

5. Synchronize Reaction To Transiently Enrich Intermediates S P I2I2 S I1I1 I2I2 I1I1 I2I2 P Molecular fate suggested by temporal order time Single molecule analyses follow this strategy but - do not require synchronization - do establish molecular fate Block Reaction Step To Accumulate Intermediate S P I1I1 I2I2 I1I1 P Examples of blocks: - remove/inactivate protein - remove cofactor - lower temperature - add inhibitor { Partial Reaction Pulse-Chase Label a Synchronous Cohort S P I1I1 I2I2 Label can sensitivity of detection Molecular fate established by chase S P I1I1 S I1I1 I2I2 I2I2 P I2I2 P S P I2I2 I1I1 I1I1 time Molecular fate suggested by block and possibly established if block can be reversed S

6. Detecting Intermediates Pulse-Chase Label a Synchronous Cohort S P I1I1 I2I2 Label can sensitivity of detection Molecular fate established by chase S P I1I1 I2I2 I2I2 S I1I1 I2I2 P P S P I2I2 I1I1 I1I1 time Synchronize Reaction To Transiently Enrich Intermediates S P I2I2 S I1I1 I2I2 I1I1 I2I2 P Molecular fate suggested by temporal order time Single molecule analyses follow this strategy but - do not require synchronization - do establish molecular fate Block Reaction Step To Accumulate Intermediate S P I1I1 I2I2 I1I1 P Examples of blocks: - remove/inactivate protein - remove cofactor - lower temperature - add inhibitor { Partial Reaction Molecular fate suggested by block and possibly established if block can be reversed S

7. Detecting Intermediates Block Reaction Step To Accumulate Intermediate S P I1I1 I2I2 I1I1 P Examples of blocks: - remove/inactivate protein - remove cofactor - lower temperature - add inhibitor { Partial Reaction Synchronize Reaction To Transiently Enrich Intermediates Pulse-Chase Label a Synchronous Cohort S S P I1I1 I2I2 P I2I2 S I1I1 I2I2 I1I1 I2I2 P Label can sensitivity of detection Molecular fate suggested by temporal order Molecular fate established by chase time S P I1I1 S I1I1 I2I2 I2I2 P I2I2 P S P I2I2 I1I1 I1I1 Single molecule analyses follow this strategy but - do not require synchronization - do establish molecular fate Molecular fate suggested by block and possibly established if block can be reversed S

8. Understanding Molecular Mechanisms SP S = substrate P = product I = intermediate A = activity How to structurally characterize intermediates? How to detect and identify intermediates? How to identify the proteins/nucleic acids responsible for the activities? SP I1I1 I2I2 I3I3 I 4………….. I n A1A1 A2A2 A3A3 A4A4 A n+1

9. Nucleic Acids Structural Analysis of Intermediates Complexes Size Shape DS versus SS Topology Modification Covalent Linkages Proteins Modification Ligand Binding Conformation Covalent Linkages Composition Stoichiometry Conformation Interacting Sequences Strand Pairing Examples of structural features that can be monitored Cofactor (NTP) Status Interacting Domains Strand Polarity

10. Structural Analysis of In Vivo DNA Replication Intermediates fork daughter parent EM can distinguish SS from DS DNA small alkaline sucrose gradient (size) SS DNA (lagging) SS DNA (lagging) DS DNA (leading) DS DNA (leading) Shape Size DS vs SS

11. Nucleic Acids Structural Analysis of Intermediates Complexes Size Shape DS versus SS Topology Modification Covalent Linkages Proteins Modification Ligand Binding Conformation Covalent Linkages Composition Stoichiometry Conformation Interacting Sequences Strand Pairing Examples of structural features that can be monitored Cofactor (NTP) Status Interacting Domains Strand Polarity

12. Understanding Molecular Mechanisms SP S = substrate P = product I = intermediate A = activity How to structurally characterize intermediates? How to detect and identify intermediates? How to identify the proteins/nucleic acids responsible for the activities? SP I1I1 I2I2 I3I3 I 4………….. I n A1A1 A2A2 A3A3 A4A4 A n+1

13. SP S = substrate P = product I = intermediate A = activity How to structurally characterize intermediates? How to detect and identify intermediates? How to identify the proteins/nucleic acids responsible for the activities? SP I1I1 I2I2 I3I3 I 4………….. I n A1A1 A2A2 A3A3 A4A4 A n+1 Advantages of Studying Molecular Mechanisms in vitro Can study a process independent of other competing or disruptive processes Easier to synchronize or pulse-label the process Easier to block steps or introduce defined intermediates Easier to isolate and structurally probe intermediates Can separate and purify activities required for a process

14. Purifying Biochemical Activities from In Vitro Systems Fractionation & ReconstitutionIn vitro complementation Can accelerate by trying to replace fractions with suspected proteins purified in expression systems

15. SP S = substrate P = product I = intermediate A = activity How to structurally characterize intermediates? How to detect and identify intermediates? How to identify the proteins/nucleic acids responsible for the activities? SP I1I1 I2I2 I3I3 I 4………….. I n A1A1 A2A2 A3A3 A4A4 A n+1 Advantages of Studying Molecular Mechanisms in vitro Can study a process independent of other competing or disruptive processes Easier to synchronize or pulse-label the process Easier to block steps or introduce defined intermediates Easier to isolate and structurally probe intermediates Can separate and purify activities required for a process

16. Validating an in vitro system Show the in vitro system shares many properties of the in vivo process Substrate Product Intermediates Genetic Requirements Inhibitor Sensitivity Quantitative Properties Example: replication elongation DS DNA template; dNTP replication fork okazaki fragment replication mutants aphidicolin (for eukaryotes) fork rate okazaki fragment size

17. Phage T4 DNA Replication In Vitro Fork Rate Okazaki Fragment Genetic Requirements in vivoin vitro 800 nt/sec500 nt/sec ~ 2 kb No OF maturation 32, 41, 43, 44, 45, 62

18. Phage T4 DNA Replication In Vitro Fork Rate Okazaki Fragment Genetic Requirements in vivoin vitro 800 nt/sec500 nt/sec ~ 2 kb No OF maturation 32, 41, 43, 44, 45, 62 Biochemical activities mostly purified by in vitro complementation Can reconstitute reaction with seven purified proteins

19. Synchronize Reaction To Transiently Enrich Intermediates The Challenge: Intermediates are often transient, scarce, and coexist with other molecular species Detecting Intermediates Pulse-Chase Label Synchronous Cohort Block Reaction Step To Accumulate Intermediate S S P I1I1 I2I2 S I1I1 S P I1I1 I2I2 S P I2I2 S P I1I1 S I1I1 I2I2 I1I1 I1I1 I2I2 I2I2 PP I1I1 I2I2 P I2I2 I2I2 P S P I1I1 I2I2 P I1I1 Label can sensitivity of detection Examples of blocks: Remove or inactivate protein Remove cofactor Lower temperature Add inhibitor Chasing into final product confirms labeled species is true intermediate Can provide temporal order of intermediates Can provide temporal order of intermediates { Partial Reaction

20. A Helix Unwinding (Helicase) Activity 41 is required for rapid strand displacement synthesis on DS DNA 41 is NOT required for rapid synthesis on SS DNA 41 has GTP/ATPase activity GTP/ATPase greatly stimulated by SS DNA Inhibition by GTP  S slows strand displacement synthesis A direct assay for helicase activity * *

21. Replicative Helicases Many replicative helicases form hexameric rings that are thought to wrap around single-stranded DNA and use the energy of nucleotide hydrolysis to translocate unidirectionally along the DNA, peeling away any hybridized DNA it runs into. 5’ 3’ 5’ 3’ 5’ 3’ 5’ The presumed translocation direction relative to the polarity of the single-stranded DNA defines the polarity of the helicase. Prokaryote: 5’ > 3’ (as modeled above on lagging strand). Eukaryotes: 3’ > 5’ (would be modeled on leading strand). These helicases belong to a family of AAA+ ATPases that form multimeric complexes and can couple ATP binding and/or hydrolysis to conformational changes in the complex

22. Okazaki Fragment Maturation Excise Primer Fill-In Gap Seal Nick Phage T4 T4 RNaseH T4 DNA Pol T4 DNA Ligase E. coli DNA Pol I (5’ > 3’ Exo) DNA Pol I DNA Ligase E. Coli OF Processing Addition of T4 RNaseH and T4 DNA Ligase to in vitro replication system produces continuous lagging strand T4 RNaseH or T4 DNA Ligase mutants accumulate okazaki fragments during phage T4 infection in vivo

23. Replication Fork Tasks and the Activities That Perform Them separate parental strands begin DNA synthesis stabilize SS DNA synthesize DNA ensure processivity unlink parental strands Task Activity helicase primase SSBP polymerase clamp loader/clamp topoisomerase connect okazaki fragments replace primer ligase nuclease/polymerase

24. Understanding Molecular Mechanisms SP S = substrate P = product I = intermediate A = activity How to structurally characterize intermediates? How to detect and identify intermediates? How to identify the proteins/nucleic acids responsible for the activities? SP I1I1 I2I2 I3I3 I 4………….. I n A1A1 A2A2 A3A3 A4A4 A n+1 Some activities may affect the rate, fidelity, specificity, or regulation of these steps How is order, timing, specificity/fidelity, and speed/efficiency maintained?

25. Processivity How many times an enzyme can act on a substrate before dissociating from it Assay: measure product size under conditions where an enzyme cannot reassociate with its substrate once it dissociates Condition 1: preload enzymes onto substrates then dilute Condition 2: excess substrate (e.g. primer-template) polymerase not processivepolymerase processive

26. An Activity that Enhances Polymerase Processivity 44/62 ATPase and 45 enhance the processivity of T4 DNA polymerase 43 Continuous ATP hydrolysis by 44/62 is not required for enhanced processivity Once ATP is hydrolyzed, processivity factors act like a “sliding clamp” for the polymerase

27. The Sliding Clamp is a ring that tethers the Polymerase

28. E. Coli Clamp-Loader (   ’) loads the Clamp (  ) onto DNA through the ordered execution of activities, each of which is dependent on the intermediate generated by the previous activity 3 Clamp Loading Model 2

29. Summary of Activities and Proteins at the Replication Fork Diagram shows prokaryotic 5’>3’ helicase on lagging strand 3’>5’ eukaryotic helicase would be placed on leading strand Task Activity E. coliEukaryotes unwind parental strandshelicase DnaB)Mcm2-7, Cdc45, GINS prime DNA synthesisprimase DNA Pol  -primase stabilize SS DNASSBP RPA1-3 synthesize DNApolymerase DNA Pol III core DNA Pol , DNA Pol  ensure processivityclamp loader, clamp  -complex,  subunit RFC1-5, PCNA unlink parental strandstopoisomeraseTopo I/Gyrase, Topo IVTopo I/Topo II connect okazaki fragmentsligase DNA LigaseDNA Ligase I replace primerDNA Pol I/RNaseH DNA Pol , FenI, Dna2 polymerase/nuclease coord leading and lagging  subunit Ctf4? ? * * DNA Pol III Holoenzyme ** **  leading,  lagging Note: Many of these activities are also required for DNA repair or recombination, and in several cases the same proteins are used

30. The Challenge of Regulating and Coordinating Multiple Activities Primase synthesizes primer Clamp-loader positions clamp around primer-template Polymerase dissociates from clamp to load onto next primer Polymerase loads onto primer-template and binds to clamp Polymerase synthesizes okazaki fragment Okazaki fragment maturation is completed Clamp-loader eventually releases clamp for reuse on other okazaki fragments Primase synthesizes primer for next okazaki fragment Clamp-loader loads clamp Adapted from Molecular Biology of the Cell. 4th Ed. Collision Model versus Primer Signaling Model What regulates polymerase processivity? What regulates primer synthesis? What directs when clamps are released?

31. Keeping the Lagging Strand Polymerase at the Replication Fork Figures from Molecular Biology of the Cell. 4th Ed. Processive synthesis of okazaki fragments by lagging strand polymerase suggests tethering to leading strand replication proteins at the fork, generating a dynamic lagging strand loop (trombone model). In E. coli, tau dimer tethers by binding two core polymerases in the Pol III holoenzyme Pol III holoenzyme core  Complex clamp-loader  clamp  dimer Predicted lagging strand “loop” seen in EM; dynamic loop behavior detected by single molecule analysis  clamp

32. Trombone Model from Cell Snapshots (Cell 141:1088) See Movie on Bioreg Website Links Page How do primase and helicase interact yet work in opposite directions? Are leading and lagging polymerization coordinated? What holds leading and lagging strand polymerases together in other systems? How many polymerases can interact with each clamp?

33. Segurado & Tercero, Biol. Cell (2009) 11:617-627 DNA lesions induce responses to: (1) protect stalled forks (2) bypass lesions (3) delay further initiation (4) block cell cycle Keeping the Fork Going Through Thick and Thin Many genomic insults are now thought to originate from replication accidents 1 2 3 4

34. DNA replication is coordinated with other cellular processes Replication of chromatin and chromatin states -- nucleosome structure and chromatin states are duplicated along with the DNA -- changes in chromatin states are associated with changes in replication timing Sister Chromatid Cohesion -- establishment of cohesion is coupled to DNA replication Meiotic Recombination -- the DS breaks that initiate meiotic recombination are coupled to DNA replication Transcription -- replication forks can survive collisions with the transcription machinery