REPLICATIONREPLICATION

Chapter 7Chapter 7

The ProblemThe Problem

DNA is maintained in a compressed, DNA is maintained in a compressed, supercoiled state.supercoiled state.

BUT, basis of replication is the formation BUT, basis of replication is the formation of strands based on specific bases of strands based on specific bases pairing with their complementary bases.pairing with their complementary bases.

Before DNA can be replicated it must Before DNA can be replicated it must be made accessible, i.e., it must be be made accessible, i.e., it must be unwound unwound

THREE HYPOTHESES FOR DNA REPLICATION

Models of ReplicationModels of Replication

(a) Hypothesis 1:

Semi-conservative replication

(b) Hypothesis 2:Conservative replication

Intermediate molecule

(c) Hypothesis 3:Dispersive replication

MODELS OF DNA REPLICATION

PREDICTED DENSITIES OFNEWLY REPLICATED DNAMOLECULES ACCORDINGTO THE THREE HYPOTHESESABOUT DNA REPLICATION

Meselson and Stahl Conclusion: Semi-conservative replication of DNA

Replication as a processReplication as a process Double-stranded DNA unwinds.Double-stranded DNA unwinds.

The junction of the unwound

molecules is a replication fork.

A new strand is formed by pairing complementary bases with theold strand.

Two molecules are made.

Each has one new and one old

DNA strand.

Extending the ChainExtending the Chain dNTPs are added individuallydNTPs are added individually Sequence determined by pairing with Sequence determined by pairing with

template strandtemplate strand DNA has only one phosphate DNA has only one phosphate

between bases, so why use dNTPs?between bases, so why use dNTPs?

Extending the ChainExtending the Chain

DNA SynthesisDNA Synthesis

2 phosphates

3’-OH nucleophilic attack on alpha phosphate of incoming dNTP

removal and splitting of pyrophosphate by inorganic pyrophosphatase

Chain Elongation in the 5’ 3’ direction

Semi-discontinuous Semi-discontinuous ReplicationReplication

All known DNA pols work in a 5’>>3’ All known DNA pols work in a 5’>>3’ directiondirection

Solution?Solution? Okazaki fragmentsOkazaki fragments

Okazaki ExperimentOkazaki Experiment

Continuous synthesis

Discontinuous synthesis

DNA replication is semi-discontinuous

Features of DNA Replication

DNA replication is DNA replication is semiconservativesemiconservative Each strand of template DNA is being copied.Each strand of template DNA is being copied.

DNA replication is DNA replication is semidiscontinuoussemidiscontinuous The leading strand copies continuouslyThe leading strand copies continuously The lagging strand copies in segments The lagging strand copies in segments

((Okazaki fragmentsOkazaki fragments) which must be joined) which must be joined DNA replication is DNA replication is bidirectionalbidirectional

Bidirectional replication involves two Bidirectional replication involves two replication forks, which move in opposite replication forks, which move in opposite directionsdirections

DNA Replication-DNA Replication-Prokaryotes Prokaryotes

DNA replication is semiconservative. DNA replication is semiconservative. the helix must be unwound.the helix must be unwound.

Most naturally occurring DNA is Most naturally occurring DNA is slightly negatively supercoiled.slightly negatively supercoiled.

Torsional strain must be released Torsional strain must be released Replication induces positive Replication induces positive

supercoilingsupercoiling Torsional strain must be released, Torsional strain must be released,

again.again. SOLUTION: TopoisomerasesSOLUTION: Topoisomerases

The Problem of OverwindingThe Problem of Overwinding

Topoisomerase Type I Topoisomerase Type I

Precedes replicating DNAPrecedes replicating DNA MechanismMechanism

Makes a cut in one strand, passes Makes a cut in one strand, passes other strand through it. Seals gap.other strand through it. Seals gap.

Result: induces positive supercoiling Result: induces positive supercoiling as strands are separated, allowing as strands are separated, allowing replication machinery to proceed. replication machinery to proceed.

HelicaseHelicase Operates in Operates in

replication forkreplication fork Separates strands to Separates strands to

allow DNA Pol to allow DNA Pol to function on single function on single strands. strands.

Translocate along single strain in 5’->3’ or 3’-> 5’ direction by hydrolyzing ATP

Gyrase--A Type II Gyrase--A Type II TopoisomeraseTopoisomerase

Introduces negative supercoilsIntroduces negative supercoils Cuts both strandsCuts both strands Section located away from actual cut Section located away from actual cut

is then passed through cut site. is then passed through cut site.

Initiation of ReplicationInitiation of Replication

Replication initiated at specific sites: Replication initiated at specific sites: Origin of Replication (Origin of Replication (oriori))

Two Types of initiation:Two Types of initiation: De novo –De novo –Synthesis initiated with RNA Synthesis initiated with RNA

primers. Most common.primers. Most common. Covalent extension—synthesis of new Covalent extension—synthesis of new

strand as an extension of an old strand strand as an extension of an old strand (“Rolling Circle”)(“Rolling Circle”)

De novoDe novo Initiation Initiation Binding to Binding to

Ori COri C by by DnaA proteinDnaA protein

Opens Opens StrandsStrands

Replication Replication proceeds proceeds bidirectionallbidirectionallyy

Unwinding the DNA by Helicase Unwinding the DNA by Helicase (DnaB protein)(DnaB protein)

Uses ATP to separate the DNA strandsUses ATP to separate the DNA strands At least 4 helicases have been At least 4 helicases have been

identified in E. coli.identified in E. coli. How was DnaB identified as the How was DnaB identified as the

helicase necessary for replication?helicase necessary for replication? NOTE: Mutation in such an essential NOTE: Mutation in such an essential

gene would be lethal.gene would be lethal. Solution?Solution?

Conditional mutantsConditional mutants

Liebowitz ExperimentLiebowitz Experiment

What would you expect if the substrates are separated by electrophoresis after treatment with a helicase?

Liebowitz Assay--ResultsLiebowitz Assay--Results What do these What do these

results indicate?results indicate? ALTHOUGH ALTHOUGH

PRIMASE (DnaG) PRIMASE (DnaG) AND SINGLE- AND SINGLE- STRAND BINDING STRAND BINDING PROTEIN (SSB) PROTEIN (SSB) BOTH STIMULATE BOTH STIMULATE DNA HELICASE DNA HELICASE (DnaB), NEITHER (DnaB), NEITHER HAVE HELICASE HAVE HELICASE ACTIVITY OF THEIR ACTIVITY OF THEIR OWNOWN

Single Stranded DNA Binding Single Stranded DNA Binding Proteins (SSB)Proteins (SSB)

Maintain strand separation once Maintain strand separation once helicase separates strandshelicase separates strands

Not only separate and protect ssDNA, Not only separate and protect ssDNA, also stimulates binding by DNA pol also stimulates binding by DNA pol (too much SSB inhibits DNA (too much SSB inhibits DNA synthesis)synthesis)

Strand growth proceeds 5’>>3’Strand growth proceeds 5’>>3’

Replication: The OverviewReplication: The Overview Requirements:Requirements:

DeoxyribonucleotidesDeoxyribonucleotides DNA templateDNA template DNA PolymeraseDNA Polymerase

5 DNA pols in E. coli5 DNA pols in E. coli 5 DNA pols in mammals5 DNA pols in mammals

PrimerPrimer ProofreadingProofreading

A total of 5 different DNAPs have been reported in E. coli

DNAP I: functions in repair and replication DNAP II: functions in DNA repair (proven in 1999)

DNAP III: principal DNA replication enzyme DNAP IV: functions in DNA repair (discovered in

1999)

DNAP V: functions in DNA repair (discovered in 1999)

To date, a total of 14 different DNA polymerases have been reported in eukaryotes

The DNA Polymerase Family

DNA pol IDNA pol I First DNA pol discovered.First DNA pol discovered. Proteolysis yields 2 chainsProteolysis yields 2 chains

Larger Chain (Klenow Fragment) 68 kdLarger Chain (Klenow Fragment) 68 kdC-terminal 2/3rd. 5’>>3’ C-terminal 2/3rd. 5’>>3’ polymerizing activitypolymerizing activity

N-terminal 1/3rd. 3’>>5’ N-terminal 1/3rd. 3’>>5’ exonuclease activityexonuclease activity

Smaller chain: 5’>>3 exonucleolytic Smaller chain: 5’>>3 exonucleolytic activityactivitynt removal 5’>>3’nt removal 5’>>3’Can remove >1 ntCan remove >1 ntCan remove deoxyribos or ribosCan remove deoxyribos or ribos

DNA pol IDNA pol I First DNA pol discovered.First DNA pol discovered. Proteolysis yields 2 chainsProteolysis yields 2 chains

Larger Chain (Klenow Fragment) 68 kdLarger Chain (Klenow Fragment) 68 kdC-terminal 2/3rd. 5’>>3’ C-terminal 2/3rd. 5’>>3’ polymerizing activitypolymerizing activity

N-terminal 1/3rd. 3’>>5’ N-terminal 1/3rd. 3’>>5’ exonuclease activityexonuclease activity

Smaller chain: 5’>>3 exonucleolytic Smaller chain: 5’>>3 exonucleolytic activityactivitynt removal 5’>>3’nt removal 5’>>3’Can remove >1 ntCan remove >1 ntCan remove deoxyribos or ribosCan remove deoxyribos or ribos

The structure of theKlenow fragment ofDNAP I from E. coli

Requires 5’-3’ activity of DNA pol I

Steps1. At a nick (free 3’ OH) in the DNA

the DNA pol I binds and digests nucleotides in a 5’-3’ direction

2. The DNA polymerase activity synthesizes a new DNA strand

3. A nick remains as the DNA pol I dissociates from the ds DNA.

4. The nick is closed via DNA ligase

Nick TranslationNick Translation

Source: Lehninger pg. 940

5'-exonuclease activity, working together with the 5'-exonuclease activity, working together with the polymerase, accomplishes "nick translation"polymerase, accomplishes "nick translation"

This activity is critical in primer removal

Nick Translation 2Nick Translation 2

DNA Polymerase I is great, DNA Polymerase I is great, but…. but….

In 1969 John Cairns and Paula deLuciaIn 1969 John Cairns and Paula deLucia -isolated a mutant bacterial strain with -isolated a mutant bacterial strain with

only 1% DNAP I activity (polA)only 1% DNAP I activity (polA)- mutant was super sensitive to UV - mutant was super sensitive to UV

radiationradiation- but otherwise the mutant was fine i.e. it - but otherwise the mutant was fine i.e. it

could could divide, so obviously it can divide, so obviously it can replicate its DNAreplicate its DNA

Conclusion: Conclusion: DNAP I is NOT the principal replication DNAP I is NOT the principal replication

enzyme in E. colienzyme in E. coli

- DNAP I is too slow (600 dNTPs - DNAP I is too slow (600 dNTPs added/minute)added/minute)

- DNAP I is only moderately processive- DNAP I is only moderately processive

(processivity refers to the number of dNTPs (processivity refers to the number of dNTPs added to a growing DNA chain before the added to a growing DNA chain before the enzyme dissociates from the template)enzyme dissociates from the template)

Conclusion:Conclusion: There must be additional DNA polymerases.There must be additional DNA polymerases. Biochemists purified them from the polA Biochemists purified them from the polA

mutantmutant

Other clues…. Other clues….

The major replicative polymerase in E. coli ~ 1,000 dNTPs added/sec It’s highly processive: >500,000 dNTPs

added before dissociating Accuracy:

1 error in 107 dNTPs added, with proofreading final error rate of 1 in

1010 overall.

DNA Polymerase III DNA Polymerase III

The 10 subunits of E. coli DNA polymerase III

Subunit Function

’

5’ to 3’ polymerizing activity3’ to 5’ exonuclease activity and assembly (scaffold)Assembly of holoenzyme on DNASliding clamp = processivity factorClamp-loading complexClamp-loading complexClamp-loading complexClamp-loading complexClamp-loading complex

Coreenzyme

Ho

loen

zym

e

DNA Polymerase III Holoenzyme DNA Polymerase III Holoenzyme (Replicase)(Replicase)

Activities of DNA Pol III ~900 kd Synthesizes both leading and

lagging strand Can only extend from a primer

(either RNA or DNA), not initiate 5’>>3’ polymerizing activity 3’>>5’ exonuclease activity NO 5’>>3’ exonuclease activity

Subsequenthydrolysis ofPPi drives thereaction forward

Nucleotides are added at the 3'-end of the strand

The 5’ to 3’ DNA polymerizing The 5’ to 3’ DNA polymerizing activity activity

Leading and Lagging Leading and Lagging StrandsStrands

REMEMBER: DNA polymerases REMEMBER: DNA polymerases require a primer.require a primer.

Most living things use an RNA primerMost living things use an RNA primer Leading strand (continuous): primer Leading strand (continuous): primer

made by RNA polymerasemade by RNA polymerase Lagging strand (discontinuous): Lagging strand (discontinuous):

Primer made by PrimasePrimer made by Primase Priming occurs near replication fork, Priming occurs near replication fork,

need to unwind helix. SOLUTION: need to unwind helix. SOLUTION: HelicaseHelicase

Primosome= Primase + HelicasePrimosome= Primase + Helicase

The The ReplisomeReplisome DNA pol III extends DNA pol III extends

on both the leading on both the leading and lagging strandand lagging strand

Growth stops when Growth stops when Pol III encounters an Pol III encounters an RNA primer (no RNA primer (no 5’>>3’ exonuclease 5’>>3’ exonuclease activity)activity)

Pol I then extends the Pol I then extends the chain while removing chain while removing the primer (5’>>3’)the primer (5’>>3’)

Stops when nick is Stops when nick is sealed by ligasesealed by ligase

Ligase Ligase Uses NADUses NAD++ or ATP for or ATP for

coupled reactioncoupled reaction 3-step reaction:3-step reaction:

AMP is transferred to Lysine AMP is transferred to Lysine residue on enzymeresidue on enzyme

AMP transferred to open 5’ AMP transferred to open 5’ phosphate via temporary phosphate via temporary pyrophosphatepyrophosphate

AMP released, AMP released, phosphodiester linkage phosphodiester linkage mademade

NADNADNMN + AMPNMN + AMP ATP ATP ADP + PPiADP + PPi

DNA Replication ModelDNA Replication Model1.1. Relaxation of supercoiled Relaxation of supercoiled

DNA.DNA.2.2. Denaturation and Denaturation and

untwisting of the double untwisting of the double helix.helix.

3.3. Stabilization of the ssDNA Stabilization of the ssDNA in the replication fork by in the replication fork by SSBs. SSBs.

4.4. Initiation of new DNA Initiation of new DNA strands. strands.

5.5. Elongation of the new DNA Elongation of the new DNA strands. strands.

6.6. Joining of the Okazaki Joining of the Okazaki fragments on the lagging fragments on the lagging strand.strand.

Termination of Termination of Replication Replication

Occurs @ specific site opposite Occurs @ specific site opposite ori cori c ~350 kb~350 kb Flanked by 6 nearly identical Flanked by 6 nearly identical non-non-

palindromic*palindromic*, 23 bp terminator (, 23 bp terminator (terter) sites) sites * Significance?* Significance?

Tus Protein-arrests replication fork motion

FIDELITY OF REPLICATIONFIDELITY OF REPLICATION Expect 1/10Expect 1/103-43-4, get 1/10, get 1/108-108-10. . FactorsFactors

3’3’5’ exonuclease activity in DNA 5’ exonuclease activity in DNA polspols

Use of “tagged” primers to initiate Use of “tagged” primers to initiate synthesissynthesis

Battery of repair enzymesBattery of repair enzymes Cells maintain balanced levels of Cells maintain balanced levels of

dNTPsdNTPs

Why Okazaki Frags?Why Okazaki Frags? Or, why not 3’Or, why not 3’5’ synthesis?5’ synthesis? Possibly due to problems with Possibly due to problems with

proofreading. proofreading. PROBLEM: PROBLEM:

Imagine a misincorporation with a 3’Imagine a misincorporation with a 3’5’ 5’ polymerasepolymerase

How is it removed?How is it removed? How is the chain extended?How is the chain extended? Is there a problem after removing a Is there a problem after removing a

mismatch?mismatch?