Transcript Bioreg2015ReplicLec2V1SlideHandout

Lecture 1:
DNA Polymerase
Use of biochemistry (assays) and genetics (phenotypes) to define function
Fidelity/Specificity: bioregulation through substrate control of molecular choice
Breaking down complex processes into intermediates and subreactions
Lecture 2:
The Replication Fork and Replisome
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
Dissecting Complex Molecular Mechanisms
S
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
S = substrate
P = product
I = intermediate
A = activity
5’
3’
3’
5’
5’
3’
Dissecting Complex Molecular Mechanisms
S
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
S = substrate
P = product
I = intermediate
A = activity
How to detect and identify intermediates?
How to structurally characterize intermediates?
How to identify the proteins/nucleic acids responsible for the activities?
Visualization of E. coli DNA Replication Intermediates
daughter
Label E. coli ~ 2 generations
with radioactive thymidine (H 3)
fork
Gently lyse cells and let DNA
settle and stick onto a membrane
parent
HL
fork
Autoradiograph with coating
of photographic emulsion
HL
HH
daughter
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
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
DNA replication is localized to two moving replication forks
that travel bidirectionally around the molecule probably from
a single site of initiation
Dissecting Complex Molecular Mechanisms
How to detect and identify intermediates?
Detecting highly abundant intermediates by precursor labeling
How to structurally characterize intermediates?
Direct visualization of single molecules by microscopy
How to identify the proteins/nucleic acids responsible for the activities?
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’
Fork Movement
3’
3’
5’
5’
3’
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
Synchronize Reaction
To Transiently Enrich
Successive Intermediates
Pulse-Chase Label a
Synchronous Cohort
time
S
S
S
I1
I2
P
S
I1
S
I1
I2
P
S
I1
I1
I2
I1
I2
P
S
I1
I2
P
I2
P
S
I1
I2
PP
Molecular fate suggested
by temporal transitions
Molecular fate established
by chase
Single molecule analyses
use similar strategy but
- do not require synchronization
- do establish molecular fate
Label can enhance sensitivity
and specificity of detection
I2
P
{
time
Block Reaction Step To
Accumulate Intermediate
Partial
Reaction
Molecular fate suggested by
block and established if
reversing block converts I to P
Examples of blocks:
- remove/inactivate protein
- remove cofactor
- lower temperature
- add inhibitor
Dissecting Complex Molecular Mechanisms
S
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
S = substrate
P = product
I = intermediate
A = activity
How to detect and identify intermediates?
How to structurally characterize intermediates?
How to identify the proteins/nucleic acids responsible for the activities?
Structural Analysis of Intermediates
Examples of structural features that can be monitored
Nucleic Acids
Proteins
Complexes
Size
Cofactor (NTP) Status
Composition
Shape
Conformation
Stoichiometry
DS versus SS
Modifications
Conformation
Strand Pairing
Ligand Binding
Interacting Sequences
Strand Polarity
Covalent Linkages
Interacting Domains
Covalent Linkages
Modifications
Topology
Sequence
Detection and Analysis of Newly Synthesized DNA
Label replicating E. coli for
seconds with H3 -thymidine
Extract DNA and alkali denature
Centrifuge in alkaline sucrose
gradient to separate by size
Measure radioactivity in
gradient fractions
(increasing size
)
The newest DNA synthesized is mostly small (~ 1000-2000 bp)
In another paper, 10-20% of the label chased into large DNA
EM visualization of fork by Inman showed SS DNA on one arm
Structural analysis by others showed 8-10 nt RNA at 5’ end
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’
D
3’Lagging
C
Fork Movement
B
A
3’
5’
Leading
5’
3’
Additional activities inferred from replication intermediate analysis
A. helix unwinding
B. priming
C. primer replacement
D. ligation
Okazaki fragment synthesis & processing
prokaryotes: 1–2 kb
eukaryotes: 100–200 bp
Using in vitro (soluble cell-free) Systems
 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
Advantages of an in vitro system to study mechanism
Can isolate a process from other competing or disruptive processes
S
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
S = substrate
P = product
I = intermediate
A = activity
How to detect and identify intermediates?
Easier to synchronize, pulse-label, or block the process
How to structurally characterize intermediates?
Easier to isolate and structurally analyze intermediates
Easier to introduce various defined intermediates (or substrates)
How to identify the proteins/nucleic acids responsible for the activities?
Can separate and purify activities without any a priori knowledge about them
Validating an in vitro system
Show the in vitro system shares many properties of the in vivo process
Example: replication elongation
Substrate
Product
Intermediates
Genetic Requirements
Inhibitor Sensitivity
Quantitative Properties
DS DNA template; dNTP
replication fork
okazaki fragment
replication mutants
aphidicolin (for eukaryotes)
fork rate
okazaki 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
in vivo
in vitro
Fork Rate
800 nt/sec
500 nt/sec
Okazaki Fragment
~ 2 kb
Genetic Requirements
32, 41, 43, 44, 45, 62
~ 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
A Helix Unwinding (Helicase) Activity
41 is required for rapid strand
displacement synthesis on DS DNA
no 41
SLOW
A direct assay for helicase activity
FAST
41 is NOT required for rapid
synthesis on SS DNA
no 41
FAST
41 has GTP/ATPase activity
Greatly stimulated by SS DNA
Inhibition by GTPS slows strand displacement
synthesis
*
*
Replicative Helicases
Form hexameric rings that encircle single-stranded DNA and
hydrolyze ATP to translocate unidirectionally along the DNA
Prokaryotes 5’ > 3’ (on lagging strand): DnaB
Eukaryotes 3’ > 5’ (on leading strand): Cdc45-Mcm2-7-GINS
Discussion
Paper
5’
5’
5’
3’
3’
3’
5’
5’
5’
DnaB
3’
3’
3’
Belong to AAA+ ATPases family, which form multimeric complexes and
couple ATP binding and/or hydrolysis to conformational changes
Activities for okazaki fragment maturation
(E. coli)
DNA Pol I (5’>3’ exo)
Excise Primer
DNA Pol I
Fill-In Gap
Ligase
Seal Nick
Replication Fork Tasks and Activities
Leading Strand
Lagging Strand
Task
Activity
synthesize DNA
polymerase
separate parental strands
helicase
prime polymerase
primase
replace primer
nuclease/polymerase
connect okazaki fragments
ligase
stabilize SS DNA
SSBP
ensure processivity
clamp loader/clamp
unlink parental strands
topoisomerase
Understanding Molecular Mechanisms
Some activities may affect the rate, fidelity, specificity, or regulation of these steps
S
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
S = substrate
P = product
I = intermediate
A = activity
How to detect and identify intermediates?
How to structurally characterize intermediates?
How to identify the proteins/nucleic acids responsible for the activities?
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
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
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
S = substrate
P = product
I = intermediate
A = activity
How to detect and identify intermediates?
How to structurally characterize intermediates?
How to identify the proteins/nucleic acids responsible for the activities?
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 loads onto primer-template and binds to clamp
Primase synthesizes primer
for next okazaki fragment
Polymerase synthesizes okazaki fragment
What regulates where and
when primers are made?
Clamp-loader loads clamp
Polymerase dissociates from clamp to load onto next primer
What regulates polymerase processivity?
Okazaki fragment maturation is completed
Clamp-loader eventually releases clamp
for reuse on other okazaki fragments
What directs when clamps are released?
Adapted from Molecular Biology of the Cell. 4th Ed.
Keeping the Lagging Strand Polymerase at the Replication Fork
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
B clamp
core
Complex
clamp-loader
t dimer
B clamp core
Pol III holoenzyme
Predicted lagging strand “loop” seen in EM; dynamic loop behavior detected by single molecule analysis
Figures from Molecular Biology of the Cell. 4th Ed.
Trombone Model from Cell Snapshots (Cell 141:1088)
How many polymerases can
interact with each clamp?
How do primase and
helicase interact yet
work in opposite
directions?
What holds leading and
lagging strand polymerases
together in other systems?
Are leading and lagging
polymerization coordinated?
See Movie at http://www.youtube.com/watch?v=4jtmOZaIvS0
Replication forks must deal with many problems and dangers
Many genomic insults are now thought to originate from replication accidents
DNA lesions induce responses to: (1) protect stalled forks
(2) bypass lesions
(3) delay further initiation
(4) block cell cycle
2
3
1
4
Segurado & Tercero, Biol. Cell (2009) 11:617-627
DNA replication is a major source of spontaneous mutations
Appendix Bioreg 2015
Replication Lecture 2
Full interpretation of the Cairns theta structure
daughter
D
fork
parent
HL
fork
HL
HH
daughter
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.
Modifying Okazaki’s Fully Discontinuous Synthesis Model
Okazaki: newly synthesized DNA is mostly small suggesting discontinuous replication on both strands
Inman & Schnos (1971):
Smith & Whitehouse (2012):
electron microscopy of replicating phage l DNA
SS is often seen on only one arm of each fork
In some cases interrupted by short DS segment
inactivate ligase in Saccharomyces cerevisiae
sequence small SS DNA
see opposite strand bias on either side of origins
DS
SS
DS
SS
DS
DS
SS
DS
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
Note:
Many of these activities
are also required for DNA
repair or recombination,
and in several cases the
same proteins are used
Diagram shows prokaryotic 5’>3’ helicase on lagging strand
3’>5’ eukaryotic helicase would be placed on leading strand
Activity
E. coli
Eukaryotes
unwind parental strands
helicase
DnaB
Mcm2-7, Cdc45, GINS
prime DNA synthesis
primase
primase
DNA Pol a-primase
stabilize SS DNA
SSBP
SSBP
RPA1-3
synthesize DNA
polymerase
DNA Pol III core
ensure processivity
clamp loader, clamp
-complex, b subunit
coord leading and lagging
?
t subunit
Ctf4?
unlink parental strands
topoisomerase
Topo I/Gyrase, Topo IV
Topo I/Topo II
DNA Pol I/RNaseH
DNA Pol d, FenI, Dna2
DNA Ligase
DNA Ligase I
Task
replace primer
connect okazaki fragments
polymerase/nuclease
ligase
* DNA Pol e, DNA Pol d **
* DNA Pol III Holoenzyme
RFC1-5, PCNA
** e leading, d lagging
E. Coli Clamp-Loader (3dd’) loads the Clamp (b 2) onto DNA
through the ordered execution of activities, each of which is
dependent on the intermediate generated by the previous activity
Key Interactions Order Activities
Clamp Loading Model
d alone can bind and open clamp interface
d’ binds d and blocks interaction with clamp
(sequesters d in the clamp-loader)
 has ATPase activity
ATP binding induces conformational
change in  and releases d from d’
(allows  to bind and open clamp)
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)
Energetics
Clamp opening depends on protein-ATP ( - ATP) and protein-protein (b - d) binding energies
Clamp closing depends on ATP hydrolysis