Using both Biochemistry and Genetics to understand function
DNA Replication: The Task and Challenge
Semiconservative
Duplication
Speed: very rapid duplication of every nucleotide
(ex: 6 x 109 bp in 8 hrs in humans)
Fidelity: extremely low error rate
(~1/109 nucleotide error rate)
Count: exactly two copies of every sequence per cell cycle
Regulation: coordination with other chromosomal events
(eg.mitosis, repair, recombination, transcription, chromatin packaging)
Enzymology of DNA Synthesis: DNA Polymerases
dNTP precursor
- pyrophosphate release provides energy
Instructed by single-stranded template
- senses complementarity of new nucleotide
Primer requirement*
- senses complementarity of primer
5’ > 3’ polymerization off primer*
- extension off 3’ hydroxyl
- moving 3’> 5’ on template
* enhances fidelity by allowing error correction
Assaying DNA Polymerase Activity
In principal: Monitor incorporation of radioactive nucleotide precursors ( )
into acid insoluble form (physically separable on filter)
In practice: Can be difficult to devise the right assay conditions when
you do not know the precise nature of the activity
Initial conditions used were really assaying a complex mixture of activities:
E. coli extract - source of polymerase activity but also kinase and nuclease activity
3H
Thymidine - converted to thymidine triphosphate by kinases in extract
DNA -
intended as nuclease decoy but nucleases convert to primer-template
and source of A,G,C nucleotides
First Experiment: 50 out of 1 million cpm insoluble
Ten Years Later: purify DNA Polymerase I and figure out enzyme requirements
DNA Polymerase Structure and Catalysis
Crystal structure of bacteriophage T7 DNA Polymerase
complexed with primer-template and dNTP
Two Mg++ ions positioned by conserved
acidic residues catalyze reaction
Primer
Template
Structure resembles a right hand
Rest of enzyme positions primer-template and dNTP
and ensures catalysis only occurs with proper “fit”
DNA Pol I has 3’ > 5’ Exonuclease Activity
Careful quantitative analysis of biochemical activity can suggest biological function
Exo Assay: 5’ T T T T* 3’
3’ AAAAAAAA 5’
DNA Pol I
no dTTP
*
T
5’ T T T 3’
3’ AAAAAAAA 5’
exo activity is slow relative to pol activity
exo activity is enhanced by stalling pol activity
or making 3’ end single-stranded
3’ mismatch generates both conditions
Proofread
Assay:
5’ T T T T* 3’
3’ AAAAAAAA 5’
5’ T T TC* 3’
3’ AAAAAAAA 5’
DNA Pol I
+ dTTP
DNA Pol I
+ dTTP
5’ T T T T*TT T T 3’
3’ AAAAAAAA 5’
5’ T T T T TT T T 3’
3’ AAAAAAAA 5’
mismatch specific exo activity under normal pol conditions
both pol and exo activities are sensing primer-template pairing
C*
The Polymerase and Exonuclease Activities of
Replicative DNA Polymerases Reside in Distinct Domains
2- Mode Model for Polymerase Function
Polymerase
Active Site
~ 30 Å
Exonuclease
Active Site
Polymerizing
Editing
Movement between P and E sites requires
primer-template unwinding
translocation of 3’ end
DNA Pol I is not the replicative DNA polymerase in E. coli
Illustrates importance of genetics for establishing functional relevance in cell
Use biochemical assay to screen for mutants lacking DNA polymerase activity
mutagenize
E. coli
plate
mutant E. coli
assay dNTP
incorporation
into DNA
extracts from single
mutant colonies
mutant 3473 (polA1)
has <1% wt activity
polA1 phenotypes: normal growth; repair deficient
Purification of residual polymerase activity from polA1 yields DNA Pol II and Pol III
Genetics later establishes that DNA Pol III is the primary replicative polymerase
Purification of DNA Pol III:
Different Template, Different Assay, Different Activity
Introducing the concept of holoenzymes and modular enzyme subassemblies
Fidelity Overview
Contributions to E coli
DNA Replication Fidelity
Fidelity Comparisons
Speed
DNA
Replication
500 bp/sec
(Prokaryotes)
Error
Rate
Product
Size
10-9 - 10-10
5 x 106
(E. coli)
Error rate
Intrinsic Fidelity (polym)
10-3 - 10-4
(sensing dNTP complementarity to template)
6 x 109
50 bp/sec
(Eukaryotes)
(humans)
Exonuclease Proofreading (polym)
1 x 1011
(sensing primer complementarity to template)
10-2 - 10-3
(lily)
Mismatch Repair (post polym)
RNA
Transcription
30 bp/sec
Protein
Translation
20 aa/sec
10-4
103 - 106
10-2
(sensing complementarity of two strands)
(distinguishing parental and daughter strands)
10-4
102 - 103
Overall Replication Fidelity
10-8 - 10-9
How to Distinguish Mismatch versus Correct Base Pair
Models for Polymerase Discrimination
Geometry From Crystal Structure
H-bonding (binding energetics)
Outside the active site, unpaired nucleotides
are H-bonded to H2O. Inside the active site
these H-bonds can be replaced by WC base
pairing but only incompletely replaced by
mismatch pairing
WC bp
Mismatch H bonding can also exacerbate
steric and stacking clashes (see below)
mismatch
WC bp
Steric Constraints (structure/geometry)
Imposed by enzyme’s “induced fit”,
which can test for precise base pair
geometry, proper base stacking, and
correct primer template fit.
mismatch
mismatch
Global structure of helix is not greatly perturbed
But there are:
differences in C1’ - C1’ distance and C1’ bond angles
protrusions of bases into major groove
loss of universal H acceptor positions in minor groove
Intrinsic Fidelity: Potential Base Pair Discrimination for dNTP at
Three Stages Of the DNA Polymerization Reaction Cycle
Arrow thickness roughly corresponds to rate constant
C
C
kconf
C
C
KD
E DNA N dNTP
kpol
E * DNA N dNTP
C
C
E DNA N+1 PPi
Reaction pathway for correct nucleotide
E DNA N
Reaction pathway for incorrect nucleotide
I
I
kpol
kconf
I
KD
E DNA N dNTP
I
E * DNA N dNTP
1
2
Rapid dNTP Binding
Pseudo-equilibrium
I
I
E DNA N+1 PPi
3
Slow Conformational Change
“Induced Fit”
Polymerization
Reaction
Example: T7 DNA Polymerase (Other polymerases discriminate differently at each stage)
I
KD
C
KD
C
> 8mM
~
20 µM
~ 400x
kconf
I
kconf
300 s -1
~
0.2 s
-1
~ 1500x
Rapid and Not Measured
Error Correction: Primer requirement allows kinetic discrimination
sensitive to base pairing of recently incorporated nucleotides
C
KD Arrow thickness roughly corresponds to rate constant
C
C
kpol
kconf
E * DNA N dNTP
C
C
E DNA N dNTP
E DNA N+1
C
C
E DNA N+2 PPi
Fast reaction pathways for correct primer with correct nucleotide
I
C
KD
Slow reaction pathways for incorrect primer with correct nucleotide
I
I
I
E DNA N+1 dNTP
E DNA N+1
C
I
C
kconf
I
E * DNA N+1 dNTP
1
2
Rapid dNTP Binding
Pseudo-equilibrium
Slow Conformational Change
“Induced Fit”
C
C
kpol
I
E DNA N+2 PPi
3
Polymerization
Reaction
Error Correction: Exonuclease activity allows the polymerase’s
kinetic discrimination to lead to different primer fates
Arrow thickness roughly corresponds to rate constant
C
E DNA N
Fast reaction pathways for correct primer with correct nucleotide
kexo
C
KD
C
C
kpol
kconf
E * DNA N dNTP
C
C
E DNA N dNTP
E DNA N+1
I
C
I
I
E DNA N+1 dNTP
E DNA N+1
kexo
E DNA N+2 PPi
KD
I
I
C
C
1
C
I
C
kconf
2
I
E * DNA N+1 dNTP
C
C
kpol
3
Slow reaction pathways for incorrect primer with correct nucleotide
C
E DNA N
I
E DNA N+2 PPi
Error Correction: Kinetic manipulation of molecular choice based
on complementarity of primer
Arrow thickness roughly corresponds to rate constant
C
E DNA N
exo
When a correct nucleotide is incorporated, 3’>5’ exonuclease activity is much slower
than 5’>3’ polymerase activity. Addition of the next nucleotide is kinetically favored.
pol
E
C
DNA N+1
E
I
DNA N+1
exo
C
E DNA N
C
E DNA N+2 PPi
pol
I
E DNA N+2 PPi
When an incorrect nucleotide is incorporated, disruption of the primer greatly
slows 5’>3’ polymerase activity for the next nucleotide (and slightly increases 3’>5’
exonuclease activity). Excision of the incorrect nucleotide is kinetically favored.
Error Correction: Kinetic manipulation of molecular choice based
on complementarity of primer
5’-TAGCTTC
3’-ATCGAAGCTCATG
Black arrow thickness roughly corresponds to relative rate constant
Light blue arrow thickness roughly corresponds to relative flux
exo
5’-TAGCTTCG
3’-ATCGAAGCTCATG
5’-TAGCTTCA
3’-ATCGAAGCTCATG
exo
5’-TAGCTTC
3’-ATCGAAGCTCATG
pol
pol
5’-TAGCTTCGA
3’-ATCGAAGCTCATG
5’-TAGCTTCAA
3’-ATCGAAGCTCATG
One General Strategy for Fidelity: Kinetic manipulation of
molecular choice between irreversible forward and discard pathways
Elimination
discard
The choice is ultimately determined by the relative flux of molecules
that proceed down the two competing pathways (light blue arrow)
Pathway irreversibility usually requires some chemical energy
expenditure (e.g dNTP hydrolysis), which could be coupled to either
pathway or to a reaction step preceding these pathways
forward
Cognate Substrate
Noncognate Substrate
discard
Elimination
Correct Product
forward
Incorrect Product
For each substrate, the molecular flux (and hence molecular choice) is
determined by the ratio of the forward to discard rate constants (black
arrows) for that substrate. For cognate substrates this ratio should
favor the forward reaction. For noncognate substrates, the ratio should
“flip” to favor the discard pathway.
In principal, just one or both pathways could discriminate between
cognate and noncognate substrates, i.e. change rate constants with
substrate. In practice, nature often discriminates with both.
How DNA Polymerases Check for Proper Base Pairing Geometry
Crystal Structure Evidence for “Induced Fit”
Stacking
Interaction
DNA Polymerase contacts minor groove
of primer-template
Primer
Template
Polymerase + Primer-Template
Polymerase + Primer-Template
+ dNTP
Base pair fit is “tested” before polymerization
Base pair fit is still “tested” after polymerization
Only W-C base pairs allow proper stacking
Purple: Interaction surface with DNA polymerase
Induced fit positions nucleotide, primer 3’, metal ions
Green: Universal H-bond acceptors
H-bonding with DNA polymerase
Many DNA polymerases in the cell have nonreplicative roles
Prokaryotic DNA Polymerases (E. coli)
Pol I
Pol II (Din A)
Pol III holoenzyme
Pol IV (Din B)
Pol V (UmuC, UmuD’2C)
DNA Replication (RNA primer removal); DNA repair
DNA repair
DNA Replication
DNA repair; TLS; adaptive mutagenesis
TLS (translesion synthesis)
Eukaryotic DNA Polymerases
Pol a
Pol b
Pol g
Pol d
Pol e
Pol q
Pol z
Pol l
Pol m
Pol k
Pol h
Pol i
Rev1
DNA Replication (Primer Synthesis)
Base excision repair
Mitochondrial DNA replication/repair
DNA Replication; nucleotide and base excision repair
DNA Replication; nucleotide and base excision repair
DNA crosslink repair
TLS
Meiosis-associated DNA repair
Somatic hypermutation
TLS
Error-free TLS past cyclobutane dimers
TLS, somatic hypermutation
TLS
Most of the nonreplicative polymerases have low fidelity and are error-prone
because they tolerate non-WC bp and lack 3’>5’ exo activity
Low fidelity is needed to bypass template lesions that are stalling replication
Low fidelity may be used to increase genetic variation in special circumstances
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’
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
Structural Analysis of In Vivo DNA Replication Intermediates
Replication is more complex than just DNA polymerization
Replication is localized to forks
(Autoradiograph E. coli DNA)
New DNA synthesis is small
(pulse label and size)
alkaline sucrose gradient
daughter
small
fork
Small SS gaps at forks
(EM replicating l DNA)
analysis can distinguish
SS from DS DNA
DS DNA
(leading)
SS DNA
(lagging)
SS DNA
(lagging)
DS DNA
(leading)
parent
fork
daughter
Suggests replication initiates
from single site but can’t say
that all molecules initiate
from same site
Higher resolution analysis
of okazaki fragments show
8-10nt RNA at 5’ end linked
5’ > 3’ to DNA
Antiparallel orientation
of SS DNA consistent with
presumptive leading/lagging
strand assignments
Replication Fork Tasks and the Activities That Perform Them
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
Structural “Solution” to DNA Replication
“. . . each chain acts as a template
for the formation on to itself
of a new companion chain.”
- Watson & Crick
20 Å
34 Å
Structural Implications
1. Complementarity semiconservative model
2. Antiparallel strandspotential asymmetry if replication
proceeds along helix in one direction
3. Double helix  must disentangle interlinked parental
strands during semiconservative replication
ref 1
Demonstrating 5’3’ Chain Growth
IF 5’3’ correct
polymer growth
+
P-P-P
P
P
P
5’
+
P-P-P
OH
3’
OH
P-P-P
P
P
P
P
5’
+
+
P-P-P
OH
OH
3’
polymer growth
IF 3’5’ correct
P-P-P
PPi
5’
P
P
P
OH
3’
P-P-P
P
P
P
P
5’
PPi
OH
3’
Test: Use dideoxy nucleotide to ask if 3’ OH required for monomer incorporation?
Result: Dideoxy is incorporated but subsequent polymerization is blocked
Polymerization via Head Growth vs Tail Growth
Head Growth: activated high energy bond end of polymer drives polymerization
Tail Growth: activated high energy bond end of monomer drives polymerization
5’>3’ allows “discard” exo pathway to return to polymerization
5’ > 3’ polymerization (head growth)
3’>5’ Exo
3’ > 5’ polymerization (hypothetical tail growth)
5’>3’ Exo
Using Biochemical Assays to Define Biochemical Functions
Assay must distinguish or physically separate products from substrates
Polymerization: conversion of radioactive nucleotide from acid soluble to acid precipitable
Nuclease: conversion of incorporated radioactive nucleotide from acid precipitable to acid soluble
Quantification is important for inferring biological relevance
3’ > 5’ exonuclease negates the polymerization reaction, but is generally much slower
DNA Pol I’s poor polymerization raised the possibility that it was not the replicative helicase
Small differences in assay conditions can define different activities
gapped/nicked template defines Pol III core activity
primed single-stranded template defines Pol III holoenzyme activity
Complications of assaying crude extracts
(beware of wasting clean thoughts on dirty enzymes)
can be detecting multiple types of activities and be affected by multiple competing activities
can be detecting multiple similar activities
The Awesome Challenges of Genetics
polA mutant revisited
Lecture 1: polA1 mutant with <1% assayable DNA Pol1 activity have relatively normal replication
Cairns concludes DNA Pol1 is not important for DNA replication
Lecture 2: DNA Pol1 plays a role in okazaki fragment maturation, an important part of replication
What happened to the awesome power of genetics?
Caveats about gene analysis
Caveats applied to polA1 mutants
Limitations in Phenotypic Analysis
Pol I activity in living polA1 cells may be greater than that measured in
vitro (in extracts) as mutant protein may be more labile or inhibitable in
the harsher in vitro setting than in vivo.
Excess Activity/Leaky Allele
E.coli has an estimated 300 molecules of DNA Pol I, most used in DNA
repair. Fewer molecules are needed for the 2 replication forks, so the
residual activity in a polA1 mutant may be sufficient. Note, although
polA1 has an early nonsense mutation, read-through of the nonsense
codon is suspected of generating the residual Pol I activity
Redundancy
We can eliminate the first two caveats with a null mutant, but the
polA∆ mutant is still viable in minimal media (although not in rich
media, where the demands for rapid DNA replication are greater). In
this mutant Pol II or Pol III is thought to substitute (poorly but
sufficiently) for Pol I in OF maturation
With all these caveat, what is the evidence that DNA Pol1 is important for OF maturation and DNA replication?
polA12 ts mutant accumulates increased OFs at restrictive temp (similar to the ts lig4 mutant)
polA12 lig4 double mutant not only accumulates OFs but rapidly ceases DNA synthesis at restrictive temp
General Strategies for Isolating DNA Synthesis Mutants
1) Screen: assay macromolecular synthesis in vivo in ts-lethal mutants
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°
Can recycle
survivors to
further enrich
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
Lots of mutants but limited in vivo assays to characterize
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
Mismatch Repair: Correction of Replication Errors
E. coli mismatch repair
DNA- both parental strands methylated at GATC sites
daughter strand transiently unmethylated after replication
MutS - recognizes mispaired bp by susceptibility to kinking
MutH - recognizes nearby GATC
MutL - association with MutS and MutH stimulates MutH to nick
unmethylated daughter strand (basis of strand bias)
Exonuclease and helicase II, directed by MutS and MutL
excise daughter strand from nick to mispaired bp
DNA polIII, clamp, clamp-loader, and SSB synthesize
replacement DNA
MutS bound to mispaired DNA
DNA
Dam methylase eventually fully methylates GATC sites so both
strands are marked as parental for next round of replication