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
Dissecting Complex Molecular Mechanisms
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
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
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
Dissecting Complex Molecular Mechanisms
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
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
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
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
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
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
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
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