using both biochemistry and genetics to understand function
DESCRIPTION
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 10 9 bp in 8 hrs in humans). Fidelity : extremely low error rate. - PowerPoint PPT PresentationTRANSCRIPT
Using both Biochemistry and Genetics to understand function
DNA Replication: The Task and Challenge
Speed: very rapid duplication of every nucleotide
Fidelity: extremely low error rate
(ex: 6 x 109 bp in 8 hrs in humans)
(~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)
SemiconservativeDuplication
Enzymology of DNA Synthesis: DNA Polymerases
dNTP precursor
Instructed by single-stranded template
Primer requirement*
5’ > 3’ polymerization off primer*
* enhances fidelity by allowing error correction
- pyrophosphate release provides energy
- senses complementarity of new nucleotide
- extension off 3’ hydroxyl
- moving 3’> 5’ on template
- senses complementarity of primer
In principal: Monitor incorporation of radioactive nucleotide precursors ( ) into acid insoluble form (physically separable on filter)
Assaying DNA Polymerase Activity
In practice: Can be difficult to devise the right assay conditions when you do not know the precise nature of the activity
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
Initial conditions used were really assaying a complex mixture of activities:
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
Structure resembles a right hand
Two Mg++ ions positioned by conservedacidic residues catalyze reaction
Crystal structure of bacteriophage T7 DNA Polymerasecomplexed with primer-template and dNTP
Rest of enzyme positions primer-template and dNTPand ensures catalysis only occurs with proper “fit”
Primer
Template
DNA Pol I has 3’ > 5’ Exonuclease Activity
Exo Assay: TAAAAAAAA
TTT TAAAAAAAA
TTT
DNA Pol I
no dTTP
exo activity is slow relative to pol activity
exo activity is enhanced by stalling pol activity or making 3’ end single-stranded
5’3’ 5’
3’ 5’3’ 5’
3’
3’ mismatch generates both conditions
ProofreadAssay:
TAAAAAAAA
TTT TAAAAAAAA
TTDNA Pol I
+ dTTP5’3’ 5’
3’ 5’3’5’
3’
**
* T*TTTT
TAAAAAAAA
TTC TAAAAAAAA
TTDNA Pol I
+ dTTP5’3’ 5’
3’ 5’3’5’
3’
* TTTTT
C*
mismatch specific exo activity under normal pol conditionsboth pol and exo activities are sensing primer-template pairing
Careful quantitative analysis of biochemical activity can suggest biological function
The Polymerase and Exonuclease Activities ofReplicative DNA Polymerases Reside in Distinct Domains
2- Mode Model for Polymerase Function
PolymeraseActive Site
ExonucleaseActive Site
~ 30 Å
Polymerizing Editing
Movement between P and E sites requiresprimer-template unwindingtranslocation of 3’ end
mutagenize plate
E. coli mutant E. coli
extracts from singlemutant colonies
assay dNTPincorporation
into DNA
mutant 3473 (polA1)has <1% wt activity
DNA Pol I is not the replicative DNA polymerase in E. coli
Use biochemical assay to screen for mutants lacking DNA polymerase activity
Illustrates importance of genetics for establishing functional relevance in cell
polA1 phenotypes: normal growth; repair deficient
Purification of residual polymerase activity from polA1 yields DNA Pol II and Pol IIIGenetics 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
Intrinsic Fidelity (polym)
Exonuclease Proofreading (polym)
Mismatch Repair (post polym)
10-3 - 10-4
10-2 - 10-3
(sensing dNTP complementarity to template)
(sensing primer complementarity to template)
(sensing complementarity of two strands)
(distinguishing parental and daughter strands)
10-2
Overall Replication Fidelity 10-8 - 10-9
Error rate
Contributions to E coliDNA Replication FidelityFidelity Comparisons
DNAReplication
RNATranscription
ProteinTranslation
SpeedErrorRate
ProductSize
10-9 - 10-10 5 x 106
6 x 109
1 x 1011
(E. coli)
(humans)
(lily)
500 bp/sec
50 bp/sec
(Prokaryotes)
(Eukaryotes)
30 bp/sec
20 aa/sec
10-4
10-4
103 - 106
102 - 103
How to Distinguish Mismatch versus Correct Base Pair
Models for Polymerase Discrimination
Steric Constraints (structure/geometry)
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
Mismatch H bonding can also exacerbate steric and stacking clashes (see below)
Imposed by enzyme’s “induced fit”, which can test for precise base pair geometry, proper base stacking, and correct primer template fit.
Geometry From Crystal Structure
Global structure of helix is not greatly perturbedBut 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
WC bp
WC bp
mismatch
mismatch
mismatch
Intrinsic Fidelity: Potential Base Pair Discrimination for dNTP atThree Stages Of the DNA Polymerization Reaction Cycle
E DNAN
E DNAN dNTPC
E DNAN dNTP I
E DNAN dNTPC*
E DNAN dNTP I*
E DNAN+1C
PPi
E DNAN+1I
PPi
Reaction pathway for correct nucleotide
Reaction pathway for incorrect nucleotide
KDC
kconfC
kpolC
kconfI kpol
I
Rapid dNTP BindingPseudo-equilibrium
Slow Conformational Change“Induced Fit”
1 2 3
PolymerizationReaction
KDI
KDC
20 µM
> 8mM~ ~ 400x
kconfC
kconfI ~~ Rapid and Not Measured
0.2 s
300 s -1
-11500x
KDI
Example: T7 DNA Polymerase (Other polymerases discriminate differently at each stage)
Arrow thickness roughly corresponds to rate constant
Rapid dNTP BindingPseudo-equilibrium
Slow Conformational Change“Induced Fit”
PolymerizationReaction
Arrow thickness roughly corresponds to rate constant
E DNAN dNTPCE DNAN dNTPC* E DNAN+2
CPPi
KDC
kpolC
E DNAN+1C
kconfC
E DNAN+2I
PPiE DNAN+1I
kconfCI
1 2 3
dNTPCE DNAN+1I
E* dNTPCDNAN+1I polk
CI
KDCI
Fast reaction pathways for correct primer with correct nucleotide
Slow reaction pathways for incorrect primer with correct nucleotide
Error Correction: Primer requirement allows kinetic discrimination sensitive to base pairing of recently incorporated
nucleotides
Error Correction: Exonuclease activity allows the polymerase’s kinetic discrimination to lead to different primer fates
E DNAN dNTPCE DNAN dNTPC* E DNAN+2
CPPi
E DNAN+1I
Fast reaction pathways for correct primer with correct nucleotide
Slow reaction pathways for incorrect primer with correct nucleotide
KDC
kpolC
kconfCI
12 3
E DNAN+1C
dNTPCE DNAN+1I
E* dNTPCDNAN+1I
E DNAN+2I
PPi
kconfC
polkCI
KDCI
E DNANC
E DNANC
kexoI
kexo
Arrow thickness roughly corresponds to rate constant
Error Correction: Kinetic manipulation of molecular choice based on complementarity of primer
E DNAN+2C
PPi
E DNAN+1I
E DNAN+1C
E DNAN+2I
PPi
E DNANC
E DNANC
pol
exo
pol
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.
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.
Arrow thickness roughly corresponds to rate constant
Error Correction: Kinetic manipulation of molecular choice based on complementarity of primer
pol
exo
pol
exo
5’-TAGCTTCG3’-ATCGAAGCTCATG
5’-TAGCTTCGA3’-ATCGAAGCTCATG
5’-TAGCTTC3’-ATCGAAGCTCATG
5’-TAGCTTC A3’-ATCGAAGCTCATG
A5’-TAGCTTC3’-ATCGAAGCTCATG
A
5’-TAGCTTC3’-ATCGAAGCTCATG
Black arrow thickness roughly corresponds to relative rate constantLight blue arrow thickness roughly corresponds to relative flux
One General Strategy for Fidelity: Kinetic manipulation of molecular choice between irreversible forward and discard pathways
forward
discard
forward
discard
Cognate Substrate Correct Product
Noncognate Substrate Incorrect Product
Elimination
Elimination
The choice is ultimately determined by the relative flux of molecules that proceed down the two competing pathways (light blue arrow)
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.
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.
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
How DNA Polymerases Check for Proper Base Pairing Geometry
Crystal Structure Evidence for “Induced Fit”
Polymerase + Primer-Template Polymerase + Primer-Template+ dNTP
DNA Polymerase contacts minor grooveof primer-template
Template
Primer
Purple: Interaction surface with DNA polymerase
Green: Universal H-bond acceptors H-bonding with DNA polymerase
Base pair fit is still “tested” after polymerizationBase pair fit is “tested” before polymerization
StackingInteraction
Only W-C base pairs allow proper stacking
Induced fit positions nucleotide, primer 3’, metal ions
Many DNA polymerases in the cell have nonreplicative roles
Pol IPol II (Din A)Pol III holoenzymePol IV (Din B)Pol V (UmuC, UmuD’2C)
Prokaryotic DNA Polymerases (E. coli)
DNA Replication (RNA primer removal); DNA repairDNA repairDNA ReplicationDNA repair; TLS; adaptive mutagenesisTLS (translesion synthesis)
Eukaryotic DNA Polymerases
Pol Pol Pol Pol Pol Pol Pol Pol Pol Pol Pol Pol Rev1
DNA Replication (Primer Synthesis)Base excision repairMitochondrial DNA replication/repairDNA Replication; nucleotide and base excision repairDNA Replication; nucleotide and base excision repairDNA crosslink repairTLSMeiosis-associated DNA repairSomatic hypermutationTLSError-free TLS past cyclobutane dimersTLS, somatic hypermutationTLS
Most of the nonreplicative polymerases have low fidelity and are error-pronebecause they tolerate non-WC bp and lack 3’>5’ exo activity
Low fidelity is needed to bypass template lesions that are stalling replicationLow 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
Synthesis with discontinuous lagging strand pieces (okazaki fragments) requires repeated:A. primingB. replacement of primed sequenceC. ligation
5’
5’
3’
3’
5’
3’
leading
lagging
A
B
C
Fork Movement
Okazaki fragment length: prokaryotes 1000 - 2000 nt; eukaryotes 100-200 nt
Structural Analysis of In Vivo DNA Replication Intermediates
Replication is localized to forks(Autoradiograph E. coli DNA)
New DNA synthesis is small(pulse label and size)
Small SS gaps at forks(EM replicating DNA)
small
alkaline sucrose gradient
fork
fork
daughter
daughter
parent
SS DNA(lagging)
SS DNA(lagging)
DS DNA(leading)
DS DNA(leading)
Antiparallel orientationof SS DNA consistent with
presumptive leading/laggingstrand assignments
Higher resolution analysisof okazaki fragments show8-10nt RNA at 5’ end linked
5’ > 3’ to DNA
Suggests replication initiatesfrom single site but can’t say
that all molecules initiatefrom same site
analysis can distinguishSS from DS DNA
Replication is more complex than just DNA polymerization
Replication Fork Tasks and the Activities That Perform Them
unwind parental strands
begin DNA synthesis
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
Structural “Solution” to DNA Replication“. . . each chain acts as a template
for the formation on to itself of a new companion chain.”
- Watson & Crick
Structural Implications
1. Complementarity semiconservative model
2. Antiparallel strandspotential asymmetry if replication proceeds along helix in one direction
3. Double helix must disentangle interlinked parental strands during semiconservative replication
ref 1
20 Å
34 Å
Demonstrating 5’3’ Chain Growth
IF 5’3’ correct
P-P-POH
5’ 3’
+
OH5’ 3’
OH
polymer growth
+ PPi
IF 3’5’ correct
+
OH5’ 3’
PPi
polymer growth
Test: Use dideoxy nucleotide to ask if 3’ OH required for monomer incorporation?
Result: Dideoxy is incorporated but subsequent polymerization is blocked
P-P-P
P-P-PP PP
P-P-POH
5’ 3’
P PP
P-P-PP PP P
OHP PPP-P-P
P
+
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’ polymerization (head growth)
3’>5’ Exo
5’>3’ Exo
3’ > 5’ polymerization (hypothetical tail growth)
5’>3’ allows “discard” exo pathway to return to polymerization
Using Biochemical Assays to Define Biochemical Functions
Assay must distinguish or physically separate products from substrates
Small differences in assay conditions can define different activities
primed single-stranded template defines Pol III holoenzyme activity
Polymerization: conversion of radioactive nucleotide from acid soluble to acid precipitable
can be detecting multiple types of activities and be affected by multiple competing activities
Complications of assaying crude extracts(beware of wasting clean thoughts on dirty enzymes)
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
gapped/nicked template defines Pol III core activity
can be detecting multiple similar activities
DNA Pol I’s poor polymerization raised the possibility that it was not the replicative helicase
The Awesome Challenges of GeneticspolA 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 – (3H Thymine incorp.)
RNA synthesis +
Protein synthesis +
Nucleotide synthesis +
2) Selective enrichment: ts mutants that fail to incorporate poisonous
nucleotide analog during transient shift to restrictive temp
wash out5-BU 5-BU
UV
Replication mutants poorly incorporate 5-BU - survive
32° 42° 42° 32°
WT incorporate 5-bromouracil (5-BU) - UV sensitive
Can recycle survivors to
further enrich
Lo
g D
NA
syn
the
sis
generation time
shift to restrictive temperature
Slow Stop: involved in initiationongoing replication continuesnew rounds blocked
Quick Stop: involved in elongationongoing replication blocked
Lots of mutants but limited in vivo assays to characterize
Time
Mismatch Repair: Correction of Replication Errors
E. coli mismatch repair
MutS bound to mispaired DNA
MutH - recognizes nearby GATCMutL - 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
DNA
Dam methylase eventually fully methylates GATC sites so bothstrands are marked as parental for next round of replication
DNA- both parental strands methylated at GATC sites daughter strand transiently unmethylated after replication MutS - recognizes mispaired bp by susceptibility to kinking