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Lecture 1: Fidelity/Specificity: bioregulation through substrate control of molecular choice Use of biochemistry (assays) and genetics (phenotypes) to.

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Presentation on theme: "Lecture 1: Fidelity/Specificity: bioregulation through substrate control of molecular choice Use of biochemistry (assays) and genetics (phenotypes) to."— Presentation transcript:

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 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 Dissecting Complex Molecular Mechanisms S = substrate P = product I = intermediate A = activity 5’5’ 5’5’ 3’3’ 5’5’ 3’3’ 3’3’ SP SP I1I1 I2I2 I3I3 I 4………….. I n A1A1 A2A2 A3A3 A4A4 A n+1

3 Dissecting Complex 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

4 Visualization of E. coli DNA Replication Intermediates Label E. coli ~ 2 generations with radioactive thymidine (H ) 3 Gently lyse cells and let DNA settle and stick onto a membrane Autoradiograph with coating of photographic emulsion Develop emulsion and analyze DNA structures under microscope, quantifying lengths Infer double-strand labeling (HH) vs single-strand labeling (HL) from quantification of silver grain density fork HLHL HLHL HH DNA replication is localized to two moving replication forks that travel bidirectionally around the molecule probably from a single site of initiation daughter parent E. coli genome is circular and replicates with a replication bubble containing two equally long daughter arms connected at each end to the remaining parental segment

5 Dissecting Complex Molecular Mechanisms How to structurally characterize intermediates? How to detect and identify intermediates? How to identify the proteins/nucleic acids responsible for the activities? Detecting highly abundant intermediates by precursor labeling Direct visualization of single molecules by microscopy

6 Reconciling polymerase directionality with antiparallel DNA strands One strand: 5’>3’ polymerase can move continuously in same direction as replication fork Other strand: 5’>3’ polymerase must move discontinuously in opposite direction as replication fork 5’5’ 5’5’ 3’3’ 3’3’ 5’5’ 3’3’ Fork Movement Is there a transient intermediate where newly synthesized DNA is in “short” single strands? Is one of the daughter molecules single-stranded near the fork ?

7 Detecting Intermediates Pulse-Chase Label a Synchronous Cohort S P I1I1 I2I2 Label can enhance sensitivity and specificity 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 Successive Intermediates S P I2I2 S I1I1 I2I2 I1I1 I2I2 P Molecular fate suggested by temporal transitions time Single molecule analyses use similar strategy but - do not require synchronization - do establish molecular fate Block Reaction Step To Accumulate Intermediate S P I1I1 I2I2 I1I1 Examples of blocks: - remove/inactivate protein - remove cofactor - lower temperature - add inhibitor { Partial Reaction Molecular fate suggested by block and established if reversing block converts I to P S

8 Dissecting Complex 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 Size Shape DS versus SS Topology Modifications Covalent Linkages Strand Pairing Examples of structural features that can be monitored Proteins Modifications Ligand Binding Conformation Covalent Linkages Cofactor (NTP) Status Complexes Composition Stoichiometry Conformation Interacting Sequences Interacting Domains Strand Polarity Sequence

10 Detection and Analysis of Newly Synthesized DNA The newest DNA synthesized is mostly small (~ bp) Label replicating E. coli for seconds with H -thymidine 3 Extract DNA and alkali denature Centrifuge in alkaline sucrose gradient to separate by size Measure radioactivity in gradient fractions (increasing size ) In another paper, 10-20% of the label chased into large DNA Structural analysis by others showed 8-10 nt RNA at 5’ end EM visualization of fork by Inman showed SS DNA on one arm

11 Semi-Discontinuous DNA Synthesis Leading strand: polymerase moves continuously in same direction as replication fork Lagging strand: polymerase moves discontinuously in opposite direction as replication fork 5’5’ 5’5’ 3’3’ 3’3’ 5’5’ 3’3’ Leading Lagging A B C Fork Movement Additional activities inferred from replication intermediate analysis B. priming C. primer replacement D. ligation A. helix unwinding Okazaki fragment synthesis & processing prokaryotes: 1–2 kb eukaryotes: 100–200 bp D

12  The advantages of an in vitro system for understanding mechanism  How one validates an in vitro system  How one can purify the activities in the in vitro system  How one can use the purified system to understand its activities Using in vitro (soluble cell-free) Systems

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 an in vitro system to study mechanism Can isolate a process from other competing or disruptive processes Easier to synchronize, pulse-label, or block the process Easier to isolate and structurally analyze intermediates Can separate and purify activities without any a priori knowledge about them Easier to introduce various defined intermediates (or substrates)

14 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

15 Purifying biochemical activities from in vitro systems Fractionation & Reconstitution In Vitro Complementation Can accelerate by trying to replace fractions with suspected proteins purified from expression systems

16 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 activities

17 A Helix Unwinding (Helicase) Activity 41 is required for rapid strand displacement synthesis on DS DNA 41 has GTP/ATPase activity Greatly stimulated by SS DNA Inhibition by GTP  S slows strand displacement synthesis A direct assay for helicase activity * * FAST SLOW no is NOT required for rapid synthesis on SS DNA FAST no 41

18 Replicative Helicases Belong to AAA+ ATPases family, which form multimeric complexes and couple ATP binding and/or hydrolysis to conformational changes Form hexameric rings that encircle single-stranded DNA and hydrolyze ATP to translocate unidirectionally along the DNA Prokaryotes 5’ > 3’ (on lagging strand): DnaB 5’5’ 3’3’ 3’3’ 5’5’ 5’5’ 3’3’ 3’3’ 5’5’ 3’3’ 5’5’ 3’3’ 5’5’ Eukaryotes 3’ > 5’ (on leading strand): Cdc45-Mcm2-7-GINS DnaB Discussion Paper

19 Activities for okazaki fragment maturation Fill-In Gap Seal Nick (E. coli) Excise Primer DNA Pol I (5’>3’ exo) DNA Pol I Ligase

20 Replication Fork Tasks and Activities separate parental strands prime polymerase stabilize SS DNA synthesize DNA ensure processivity unlink parental strands TaskActivity helicase primase SSBP polymerase clamp loader/clamp topoisomerase connect okazaki fragments replace primer ligase nuclease/polymerase Leading Strand Lagging Strand

21 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

22 Processivity How many times an enzyme can act repeatedly 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) distributive polymerase (not processive) processive polymerase

23 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

24 The sliding clamp is a ring that tethers the polymerase

25 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 How is proper order and timing of activities maintained?

26 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. What regulates polymerase processivity? What regulates where and when primers are made? What directs when clamps are released?

27 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

28 Trombone Model from Cell Snapshots (Cell 141:1088) See Movie at 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?

29 Segurado & Tercero, Biol. Cell (2009) 11: DNA lesions induce responses to: (1) protect stalled forks (2) bypass lesions (3) delay further initiation (4) block cell cycle Replication forks must deal with many problems and dangers Many genomic insults are now thought to originate from replication accidents

30 DNA replication is a major source of spontaneous mutations

31 Appendix Bioreg 2015 Replication Lecture 2

32 D Full interpretation of the Cairns theta structure fork HLHL HLHL HH daughter parent At the time label was added the great grandparent molecule, which had initiated from an origin near the bottom left corner, had replicated all but the region from C to D (marked by arrowheads). As this round of replication was completed the resulting grandparent molecule became labeled on one strand just between C and D Initiation and completion of the next round of replication generated the parent molecule with one strand fully labeled and the other (inherited from the grandparent molecule) labeled only from C to D. Thus, the molecule is labeled on both strands between C and D and This parent molecule was then caught in the act of replicating bwith two thirds of it replicated by forks X and Y, generating two daughter arms labeled A and B. Arm A was derived from the mostly unlabeled parental strand and is thus mostly labeled only on the new daughter strand (except from D to X). Arm B was derived from the labeled parental strand and is thus labeled on both strands.

33 Inman & Schnos (1971): electron microscopy of replicating phage DNA SS is often seen on only one arm of each fork In some cases interrupted by short DS segment Modifying Okazaki’s Fully Discontinuous Synthesis Model Okazaki: newly synthesized DNA is mostly small suggesting discontinuous replication on both strands Smith & Whitehouse (2012): inactivate ligase in Saccharomyces cerevisiae sequence small SS DNA see opposite strand bias on either side of origins DS SS DS SS DS Thus, there is in vivo evidence supporting semi-discontinuous DNA synthesis (see slide notes)

34 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

35 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 Key Interactions Order Activities  alone can bind and open clamp interface  ’ binds  and blocks interaction with clamp (sequesters  in the clamp-loader) ATP binding induces conformational change in  and releases  from  ’ (allows  to bind and open clamp)  has ATPase activity Clamp binding inhibits  ATPase (prevents premature clamp release) Clamp binding enhances clamp-loader binding to primer-template ( promotes clamp delivery to DNA) Primer-template binding stimulates  ATPase (allows  to release and  close clamp to complete loading) Clamp opening depends on protein-ATP (  - ATP) and protein-protein (  -  ) binding energies Clamp closing depends on ATP hydrolysis Energetics


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