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

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

Dissecting Complex Molecular Mechanisms

S = substrate

P = product

I = intermediate

A = activity

5’

5’3’

5’

3’

3’

S PS PI1 I2 I3 I4………….. In

A1 A2 A3 A4 An+1


S P 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?

S PI1 I2 I3 I4………….. In

A1 A2 A3 A4 An+1

Visualization of E. coli DNA Replication Intermediates

Label E. coli ~ 2 generationswith radioactive thymidine (H )3

Gently lyse cells and let DNAsettle and stick onto a membrane

Autoradiograph with coatingof photographic emulsion

Develop emulsion and analyzeDNA structures under microscope,quantifying lengths

Infer double-strand labeling (HH)vs single-strand labeling (HL) fromquantification of silver grain density

fork

fork HL

HL

HH

DNA replication is localized to two moving replication forksthat travel bidirectionally around the molecule probably froma single site of initiation

daughter

daughter

parent

E. coli genome is circular and replicates with a replicationbubble containing two equally long daughter armsconnected at each end to the remaining parental segment





Detecting highly abundant intermediates by precursor labeling

Direct visualization of single molecules by microscopy

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’

3’

3’

5’

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 ?

Detecting Intermediates

Pulse-Chase Label aSynchronous Cohort

S PI1 I2

Label can enhance sensitivityand specificity of detection

Molecular fate established by chase

S PI1I2I2

S I1 I2 PP

S PI2I1I1

time

Synchronize ReactionTo Transiently Enrich

Successive Intermediates

S

PI2

S I1I2

I1 I2P

Molecular fate suggested by temporal transitions

time

Single molecule analysesuse similar strategy but

- do not require synchronization- do establish molecular fate

Block Reaction Step ToAccumulate Intermediate

S PI1 I2

I1

Examples of blocks:- remove/inactivate protein- remove cofactor- lower temperature- add inhibitor

{

PartialReaction

Molecular fate suggested by block and established if

reversing block converts I to P

S


S P S = substrate

P = product

I = intermediate

A = activity




S PI1 I2 I3 I4………….. In

A1 A2 A3 A4 An+1

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 DomainsStrand Polarity

Sequence

Detection and Analysis of Newly Synthesized DNA

The newest DNA synthesized is mostly small (~ 1000-2000 bp)

Label replicating E. coli forseconds with H -thymidine3

Extract DNA and alkali denature

Centrifuge in alkaline sucrosegradient to separate by size

Measure radioactivity ingradient 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

Semi-Discontinuous DNA SynthesisLeading strand: polymerase moves continuously in same direction as replication fork

Lagging strand: polymerase moves discontinuously in opposite direction as replication fork

5’

5’

3’

3’

5’

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 & processingprokaryotes: 1–2 kbeukaryotes: 100–200 bp

D

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

S P S = substrate

P = product

I = intermediate

A = activity




S PI1 I2 I3 I4………….. In

A1 A2 A3 A4 An+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)

Validating an in vitro systemShow 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 rateokazaki fragment size

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

Phage T4 DNA Replication in vitro

Fork Rate

Okazaki Fragment

Genetic Requirements

in vivo in vitro

800 nt/sec 500 nt/sec

~ 2 kb ~ 2 kbNo OF maturation

32, 41, 43, 44, 45, 62 32, 41, 43, 44, 45, 62

Biochemical activities mostly purified by in vitro complementationCan reconstitute reaction with seven purified activities

A Helix Unwinding (Helicase) Activity

41 is required for rapid stranddisplacement synthesis on DS DNA

41 has GTP/ATPase activity

Greatly stimulated by SS DNA

Inhibition by GTPS slows strand displacementsynthesis

A direct assay for helicase activity

**

FAST

SLOWno 41

41 is NOT required for rapidsynthesis on SS DNA

FASTno 41

Replicative Helicases

Belong to AAA+ ATPases family, which form multimeric complexes andcouple ATP binding and/or hydrolysis to conformational changes

Form hexameric rings that encircle single-stranded DNA andhydrolyze ATP to translocate unidirectionally along the DNA

Prokaryotes 5’ > 3’ (on lagging strand): DnaB

5’

3’

3’

5’

5’

3’

3’

5’

3’

5’

3’

5’

Eukaryotes 3’ > 5’ (on leading strand): Cdc45-Mcm2-7-GINS

DnaB

DiscussionPaper

Activities for okazaki fragment maturation

Fill-In Gap

Seal Nick

(E. coli)

Excise Primer

DNA Pol I (5’>3’ exo)

DNA Pol I

Ligase

Replication Fork Tasks and Activities

separate parental strands

prime polymerase

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

Leading Strand

Lagging Strand

Understanding Molecular Mechanisms

S P S = substrate

P = product

I = intermediate

A = activity




S PI1 I2 I3 I4………….. In

A1 A2 A3 A4 An+1

Some activities may affect the rate, fidelity, specificity, or regulation of these steps

ProcessivityHow 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)

distributivepolymerase

(not processive)

processivepolymerase

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

The sliding clamp is a ring that tethers the polymerase

Understanding Molecular Mechanisms

S P S = substrate

P = product

I = intermediate

A = activity




S PI1 I2 I3 I4………….. In

A1 A2 A3 A4 An+1

How is proper order and timing of activities maintained?

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 clampfor reuse on other okazaki fragments

Primase synthesizes primerfor 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?

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

core

Complexclamp-loader

clamp

dimer

Predicted lagging strand “loop” seen in EM; dynamic loop behavior detected by single molecule analysis

clamp

Trombone Model from Cell Snapshots (Cell 141:1088)

See Movie at http://www.youtube.com/watch?v=4jtmOZaIvS0

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?

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

Replication forks must deal with many problems and dangersMany genomic insults are now thought to originate from replication accidents

1

2

3

4

DNA replication is a major source of spontaneous mutations

Appendix Bioreg 2015Replication Lecture 2

D

Full interpretation of the Cairns theta structure

fork

fork HL

HL

HH

daughter

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.

Inman & Schnos (1971):

electron microscopy of replicating phage DNASS is often seen on only one arm of each forkIn some cases interrupted by short DS segment

Modifying Okazaki’s Fully Discontinuous Synthesis ModelOkazaki: newly synthesized DNA is mostly small suggesting discontinuous replication on both strands

Smith & Whitehouse (2012):

inactivate ligase in Saccharomyces cerevisiaesequence small SS DNAsee opposite strand bias on either side of origins

DSDS

DS

SS

SS

DS DSSS SS

DS

Thus, there is in vivo evidence supporting semi-discontinuous DNA synthesis (see slide notes)

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. coli Eukaryotes

unwind parental strands helicase DnaB Mcm2-7, Cdc45, GINS

prime DNA synthesis primase primase DNA Pol -primase

stabilize SS DNA SSBP SSBP RPA1-3

synthesize DNA polymerase DNA Pol III core DNA Pol , DNA Pol

ensure processivity clamp loader, clamp -complex, subunit RFC1-5, PCNA

unlink parental strands topoisomerase Topo I/Gyrase, Topo IV Topo I/Topo II

connect okazaki fragments ligase DNA Ligase DNA Ligase I

replace primer DNA Pol I/RNaseH DNA Pol , FenI, Dna2polymerase/nuclease

coord leading and lagging subunit Ctf4??

*

* DNA Pol III Holoenzyme

**

** leading, lagging

Note:Many of these activitiesare also required for DNArepair or recombination,and in several cases thesame proteins are used

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 andclose clamp to complete loading)

Clamp opening depends on protein-ATP ( - ATP) and protein-protein ( - ) binding energies

Clamp closing depends on ATP hydrolysis

Energetics

Documents

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