Lecture 1:
DNA Polymerase
Use of biochemistry (assays) and genetics (phenotypes) to define function
Structure critically informs but does not define function
Fidelity/Specificity: bioregulation through substrate control of molecular choice
5’
5’
5’
5’
Lecture 2:
The Replication Fork and Replisome
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
5’
3’
5’
3’
3’
5’
Semi-Discontinuous DNA Synthesis at Replication Forks
Leading daughter strand: polymerase moves continuously in same direction as replication fork
Lagging daughter strand: polymerase moves discontinuously in opposite direction as replication fork
5’
3’
Fork Movement
C
lagging
B
A
3’
5’
leading
5’
3’
Synthesis with discontinuous lagging strand pieces (okazaki fragments) requires repeated:
A. priming
B. replacement of primed sequence
C. ligation
Okazaki fragment length: prokaryotes 1000 - 2000 nt; eukaryotes 100-200 nt
Understanding Molecular Mechanisms
From This:
S
P
S = substrate
P = product
To This:
S
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
I = intermediates
A = activity
The transformation of one intermediate into another defines
activities which are performed by proteins and/or nucleic acids
How does one detect and identify intermediates?
How does one structurally characterize intermediates?
How does one identify the proteins/nucleic acids responsible for the activities?
Understanding Molecular Mechanisms
From This:
S
P
S = substrate
P = product
To This:
S
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
I = intermediates
A = activity
The transformation of one intermediate into another defines
activities which are performed by proteins and/or nucleic acids
How does one identify and detect intermediates?
How does one structurally characterize intermediates?
How does one identify the proteins/nucleic acids responsible for the activities?
Detecting Intermediates
The Challenge: Intermediates are often transient, scarce,
and coexist with other molecular species
Pulse-Chase Label
Synchronous Cohort
S
S
S
I1
I2
P
S
II11
I2
P
I2
I1
I2
P
S
I1
II22
P
I2
P
S
I1
I2
P
Single molecule analyses
follow this principal but don’t
require synchronization
Can provide temporal order
of intermediates
Label can
I2
P
Block Reaction Step To
Accumulate Intermediate
I1
Can provide temporal order
of intermediates
I1
sensitivity of detection
Chasing into final product confirms
labeled species is true intermediate
S
I1
I2
I1
P
P
{
Synchronize Reaction
To Transiently Enrich
Intermediates
S
Partial
Reaction
Examples of blocks:
Remove or inactivate protein
Remove cofactor
Lower temperature
Add inhibitor
Detecting Intermediates
The Challenge: Intermediates are often transient, scarce,
and coexist with other molecular species
Pulse-Chase Label
Synchronous Cohort
S
S
S
I1
I2
P
S
II11
I2
P
I2
I1
I2
P
S
I1
II22
P
I2
P
S
I1
I2
P
Single molecule analyses
follow this principal but don’t
require synchronization
Can provide temporal order
of intermediates
Label can
I2
P
Block Reaction Step To
Accumulate Intermediate
I1
Can provide temporal order
of intermediates
I1
sensitivity of detection
Chasing into final product confirms
labeled species is true intermediate
S
I1
I2
I1
P
P
{
Synchronize Reaction
To Transiently Enrich
Intermediates
S
Partial
Reaction
Examples of blocks:
Remove or inactivate protein
Remove cofactor
Lower temperature
Add inhibitor
Detecting Intermediates
The Challenge: Intermediates are often transient, scarce,
and coexist with other molecular species
Pulse-Chase Label
Synchronous Cohort
S
S
S
I1
I2
P
S
II11
I2
P
I2
I1
I2
P
S
I1
II22
P
I2
P
S
I1
I2
P
Single molecule analyses
follow this principal but don’t
require synchronization
Can provide temporal order
of intermediates
Label can
I2
P
Block Reaction Step To
Accumulate Intermediate
I1
Can provide temporal order
of intermediates
I1
sensitivity of detection
Chasing into final product confirms
labeled species is true intermediate
S
I1
I2
I1
P
P
{
Synchronize Reaction
To Transiently Enrich
Intermediates
S
Partial
Reaction
Examples of blocks:
Remove or inactivate protein
Remove cofactor
Lower temperature
Add inhibitor
Understanding Molecular Mechanisms
From This:
S
P
S = substrate
P = product
To This:
S
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
I = intermediates
A = activity
The transformation of one intermediate into another defines
activities which are performed by proteins and/or nucleic acids
How does one identify and detect intermediates?
How does one structurally characterize intermediates?
How does one identify the proteins/nucleic acids responsible for the activities?
Structural Analysis of Intermediates
Examples of structural features of intermediates that can be monitored
Nucleic Acids
Proteins
Complexes
Size
Cofactor (NTP) Status
Composition
Shape
Conformation
Stoichiometry
DS versus SS
Modification
Conformation
Strand Pairing
Ligand Binding
Interacting Sequences
Strand Polarity
Covalent Linkages
Interacting Domains
Covalent Linkages
Modification
Topology
Detection and Analysis of In Vivo DNA Replication Intermediates
Replication is localized to forks
(Autoradiograph E. coli DNA)
New DNA synthesis is small
(pulse label then size)
alkaline sucrose gradient
daughter
small
fork
Semi-discontinuous synthesis
(EM replicating l DNA)
EM can distinguish
SS from DS DNA
DS DNA
(leading)
SS DNA
(lagging)
SS DNA
(lagging)
DS DNA
(leading)
parent
HL
fork
HL
HH
daughter
(size)
Infer H (labeled) and L (unlabeled)
from film grain density
Short (5 - 10 sec) pulse
thought to represent initial
nucleotide incorporation
SS regions in trans, some
interrupted by small DS segments
consistent with discontinuous
okazaki fragment synthesis only on
strand of specific polarity (lagging)
Detection and Analysis of In Vivo DNA Replication Intermediates
Replication is localized to forks
(Autoradiograph E. coli DNA)
New DNA synthesis is small
(pulse label then size)
alkaline sucrose gradient
daughter
small
fork
Semi-discontinuous synthesis
(EM replicating l DNA)
EM can distinguish
SS from DS DNA
DS DNA
(leading)
SS DNA
(lagging)
SS DNA
(lagging)
DS DNA
(leading)
parent
HL
fork
HL
HH
daughter
(size)
Infer H (labeled) and L (unlabeled)
from film grain density
Short (5 - 10 sec) pulse
thought to represent initial
nucleotide incorporation
SS regions in trans, some
interrupted by small DS segments
consistent with discontinuous
okazaki fragment synthesis only on
strand of specific polarity (lagging)
Detection and Analysis of In Vivo DNA Replication Intermediates
Replication is localized to forks
(Autoradiograph E. coli DNA)
New DNA synthesis is small
(pulse label then size)
alkaline sucrose gradient
daughter
small
fork
Semi-discontinuous synthesis
(EM replicating l DNA)
EM can distinguish
SS from DS DNA
DS DNA
(leading)
SS DNA
(lagging)
SS DNA
(lagging)
DS DNA
(leading)
parent
HL
fork
HL
HH
daughter
(size)
Infer H (labeled) and L (unlabeled)
from film grain density
Short (5 - 10 sec) pulse
thought to represent initial
nucleotide incorporation
SS regions in trans, some
interrupted by small DS segments
consistent with discontinuous
okazaki fragment synthesis only on
strand of specific polarity (lagging)
DNA Synthesis Occurs Semi-Discontinuously at Replication Forks
Leading daughter strand: polymerase moves continuously in same direction as replication fork
Lagging daughter strand: polymerase moves discontinuously in opposite direction as replication fork
5’
3’
C
lagging
B
Fork Movement
A
3’
5’
leading
5’
3’
Synthesis of Okazaki fragments (1,000-2,000 nt in prokaryotes; 100-200 nt in eukaryote):
A. priming
B. replacement of primed sequence
C. ligation
Replication Fork Tasks and the Activities
Task
Activity
unwind parental strands
helicase
begin DNA synthesis
primase
stabilize SS DNA
SSBP
synthesize DNA
polymerase
ensure processivity
clamp loader/clamp
unlink parental strands
topoisomerase
replace primer
nuclease/polymerase
connect okazaki fragments
ligase
Advantages of Studying Molecular Mechanisms in vitro
Can study a process independent of other processes
From This:
S
P
S = substrate
P = product
To This:
S
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
I = intermediates
A = activity
The transformation of one intermediate into another defines
activities which are performed by proteins and/or nucleic acids
How does one identify and detect intermediates?
Easier to synchronize or pulse-label the process
Easier to block steps or introduce defined intermediates
How does one structurally characterize intermediates?
Easier to structurally probe or isolate intermediates
How does one identify the proteins/nucleic acids responsible for the activities?
Can separate and purify activities required for a process
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
DS DNA template; dNTP
replication fork
okazaki fragment
replication mutants
Inhibitor Sensitivity
aphidicolin (for eukaryotes)
Quantitative Properties
fork rate
okazaki fragment size
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
DS DNA template; dNTP
replication fork
okazaki fragment
replication mutants
Inhibitor Sensitivity
aphidicolin (for eukaryotes)
Quantitative Properties
fork rate
okazaki fragment size
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
Understanding Molecular Mechanisms
From This:
S
P
S = substrate
P = product
To This:
S
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
I = intermediates
A = activity
The transformation of one intermediate into another defines
activities which are performed by proteins and/or nucleic acids
How does one identify and detect intermediates?
How does one structurally characterize intermediates?
How does one identify the proteins/nucleic acids responsible for the activities?
Purifying Biochemical Activities from In Vitro Systems
Fractionation & Reconstitution
In vitro complementation
Can accelerate by trying to replace fractions with suspected proteins purified in 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 proteins
Detecting Intermediates
The Challenge: Intermediates are often transient, scarce,
and coexist with other molecular species
Pulse-Chase Label
Synchronous Cohort
S
S
S
I1
I2
P
S
II11
I2
P
I2
I1
I2
P
S
I1
II22
P
I2
P
S
I1
I2
P
Can provide temporal order
of intermediates
Label can
I2
P
Block Reaction Step To
Accumulate Intermediate
I1
Can provide temporal order
of intermediates
I1
sensitivity of detection
Chasing into final product confirms
labeled species is true intermediate
S
I1
I2
I1
P
P
{
Synchronize Reaction
To Transiently Enrich
Intermediates
S
Partial
Reaction
Examples of blocks:
Remove or inactivate protein
Remove cofactor
Lower temperature
Add inhibitor
A Helix Unwinding (Helicase) Activity
41 is required for rapid strand
displacement synthesis on DS DNA
A direct assay for helicase activity
*
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
*
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.
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
5’
3’
5’
3’
3’
5’
5’
3’
3’
5’
3’
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).
Okazaki Fragment Maturation
Phage T4
E. coli
Excise Primer
T4 RNaseH
DNA Pol I
(5’ > 3’ Exo)
Fill-In Gap
T4 DNA Pol
DNA Pol I
Seal Nick
T4 DNA Ligase
DNA Ligase
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
E. Coli OF Processing
Replication Fork Tasks and the Activities
Task
Activity
unwind parental strands
helicase
begin DNA synthesis
primase
stabilize SS DNA
SSBP
synthesize DNA
polymerase
ensure processivity
clamp loader/clamp
unlink parental strands
topoisomerase
replace primer
nuclease/polymerase
connect okazaki fragments
ligase
Understanding Molecular Mechanisms
From This:
S
P
S = substrate
P = product
To This:
S
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
I = intermediates
A = activity
The transformation of one intermediate into another defines
activities which are performed by proteins and/or nucleic acids
Other activities may affect the rate, fidelity, specificity, or regulation of these steps
How does one identify and detect intermediates?
How does one structurally characterize intermediates?
How does one identify the proteins/nucleic acids responsible for an activity?
How is order, timing, specificity/fidelity, and speed/efficiency maintained?
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 processive
polymerase processive
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
A clamp loader topologically links the clamp to DS DNA
Clamp Structure
The clamp also interacts with many other proteins to
coordinate their function in replication and repair
E. Coli Clamp-Loader (3 dd’) loads the Clamp (b 2) onto DNA
through the ordered execution of activities, each of which is
dependent on the substrate generated by the previous activity
Clamp Loading Model
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
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 primer synthesis?
Clamp-loader loads clamp
Polymerase dissociates from clamp to load onto next primer
What regulates polymerase processivity?
Collision Model versus
Primer Signaling Model
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.
Support for Collision Model of Lagging Strand Polymerase release
Pol III* - b clamp association is sensitive to Pol III colliding into 5’ end
all 4 dNTPs
Mimic “replicating” Pol III
by idling with only 2 dNTPs
Pol III* stably associates with
b clamp on DNA
Pol III completes replication and
runs into back of primer
Pol III* rapidly dissociates from b clamp on
DNA and within 1 sec reassociates with b
clamp preloaded on new primer-template
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 on Bioreg Website Links Page
Lecture 1:
DNA Polymerase
Use of biochemistry (assays) and genetics (phenotypes) to define function
Structure critically informs but does not define function
Fidelity/Specificity: bioregulation through substrate control of molecular choice
5’
5’
5’
5’
Lecture 2:
The Replisome
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
5’
5’
5’
5’
5’
5’
General Strategies for Isolating DNA Synthesis Mutants
1) Screen: isolate ts-lethal mutants then assay macromolecular synthesis
soon after shift to restrictive temp
DNA synthesis
RNA synthesis
Protein synthesis
Nucleotide synthesis
– (3H Thymine incorp.)
+
+
+
2) Selective enrichment: ts mutants that fail to incorporate poisonous nucleotide
analog during transient shift to restrictive temp
32°
42°
5-BU
42°
wash out
5-BU
32°
UV
WT incorporate 5-bromouracil (5-BU) - UV sensitive
Replication mutants poorly incorporate 5-BU - survive
Functional analysis of replication mutants limited by in vivo assays
Log DNA synthesis
generation time
Time
shift to restrictive temperature
Slow Stop: involved in initiation
ongoing replication continues
new rounds blocked
Quick Stop: involved in elongation
ongoing replication blocked
Assigning an activity to a protein(s)/nucleic acid(s)
What is necessary for an activity? (can be asked in vitro and in vivo)
Does absence or inactivation of a protein/nucleic acid lead to
accumulation and disappearance of the appropriate intermediates?
What is sufficient for an activity? (best asked in vitro)
Can the key feature of the activity be assayed in isolation?
Can a purified protein(s) and/or nucleic acid(s) perform that isolated activity?
An Activity that Primes DNA Synthesis (Primase)
Polymerase/primase
Coupled Assay
61
Direct Analysis
by TLC, PAGE, or RNA sequencing
41 (helicase) and 61 (primase) can act together as a mobile primosome
that has much better helicase and primase activity than either alone.
SS DNA Binding Protein helps present and protect SS DNA
Binds cooperatively to single-stranded DNA and stretches out the phosphate backbone
protein-DNA and protein-protein binding energy stabilizes
unwound DNA and makes unwinding energetically less costly
prevents secondary structure from
interfering with enzyme function
Protects sugar-phosphate backbone from nucleases while leaving bases available for pairing
RPA (eukaryotic SSBP) bound to 27 nt SS DNA
side view down axis of SS DNA
bases
accessible
Edwards lab,
CSHSQB (2000) 65:193-200
{
Topoisomerases Solve Topological Problems During Replication
Fork movement can generate superhelical tension
Daughter DNA can be left catenated after termination
converging replication forks
Two fundamental classes of topoisomerases:
Type I can transiently break single-stranded DNA and pass another single-strand through the break
Type II can transiently break double-stranded DNA and pass another double-strand through the break
Each enzyme class holds onto the broken DNA ends by reversible covalent linkage
Although not intuitively obvious, both strand passing events will relieve superhelical tension
Only Type II topoisomerases, however, can decatenate DS DNA
Type I mechanism
Type II mechanism
Cross-section of
passed strand
Figures from Wang Lab, Nat Rev Mol Cell Biol. (2002) 3:430
Advantages of In Vitro Systems for Mechanistic Studies
S
A1
I1
A2
I2
A3
I3
A4
I4………….. In
An+1
P
Isolating the Process: Can study a process independent of other processes
Identifying the Players: Can purify activities required for a process
Temporal/Kinetic Analysis: Easier to synchronize or pulse-label the process
Establishing Partial Reactions: Easier to block steps or use defined intermediates
Characterizing Intermediates: Easier to probe or isolate intermediates