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