Molecular BiologyFifth Edition

Chapter 20

DNA Replication, Damage, and Repair

Lecture PowerPoint to accompany

Robert F. Weaver

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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20.1 General Features of DNA Replication

• Double helical model for DNA includes the concept that 2 strands are complementary

• Each strand can serve as template for making its own partner– Semiconservative model for DNA replication

is correct– Half-discontinuous (short pieces later stitched

together)– Requires DNA primers– Usually bidirectional

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Three Hypotheses of Replication

The three methods of DNA replication considered were:

1. Semiconservative

2. Conservative

3. Dispersive

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• DNA replicates in a semiconservative manner

• When parental strands separate– Each strand serves as template– Makes a new, complementary strand

Semiconservative Replication

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Semidiscontinuous Replication

• DNA replication in E. coli (and in other organisms) is semidiscontinuous

• One strand (the leading strand) is replicated continuously in the direction of the movement of the replicating fork

• The other strand (the lagging strand) is replicated discontinuously as 1-2 kb Okazaki fragments in the opposite direction

• This allows both strands to be replicated in the 5’3’-direction

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DNA Replication Models

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Priming DNA Synthesis

• Okazaki fragments in E. coli are initiated with RNA primers 10-12 nt long

• Intact primers are difficult to detect in wild-type cells because of enzymes that attack RNAs

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Bidirectional Replication

• The replication structure resembles the Greek letter,

• DNA replication begins with the creation of a “bubble” – a small region where parental strands have separated and progeny DNA has been synthesized

• As the bubble expands, replicating DNA begins to take on the shape

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Theta Mode of DNA Replication in E.coli

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Replication Fork

• In DNA replication, the replication forks represent the sites of DNA replication

• Direction of replication: – Unidirectional – one fork moving away from the other

which remains fixed at the origin of replication – Bidirectional – two replicating forks moving in

opposite directions away from the origin

• Origin of replication is the fixed starting point for DNA replication

• The replicon is the DNA under the control of one origin of replication

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Rolling Circle Replication

• Circular DNAs can replicate by a rolling circle mechanism– One strand of a dsDNA is nicked and the 3’-end is

extended– This uses the intact DNA strand as a template– The 5’-end is displaced

• Phage X174 replication cycles so that when one round is complete a full-length, single-stranded circle of DNA is released

• Phage , displaced strand serves as the template for discontinuous, lagging strand synthesis

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Phage Rolling Circle Model

• As the circle rolls right– Leading strand elongates continuously

– Lagging strand elongates discontinuously• Uses unrolled leading strand as a template • RNA primers for Okazaki fragments

• Progeny dsDNA produced grows to many genomes before one genome worth is clipped off

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20.2 Enzymology of DNA Replication

• Over 30 different proteins or enzymes cooperate in replicating the E. coli DNA

• Examine the activities of some of these proteins and their homologs in other organisms– Start with DNA polymerases – the enzymes

that make DNA

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E. coli DNA Polymerases

• There are 3 DNA polymerases, the enzymes that make DNA, found in E. coli: – pol I

– pol II

– pol III

• E. coli DNA polymerase I was the first polymerase identified

• It was discovered in 1958 by Arthur Kornberg

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DNA Polymerase I

• DNA polymerase I (pol I) is a versatile enzyme with 3 distinct activities– DNA polymerase– 3’5’ exonuclease– 5’3’ exonuclease– Mild proteolytic treatment results in 2

polypeptides• Klenow fragment (the large domain)• Smaller fragment

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Klenow Fragment

Contains both: Polymerase and 3’5’ exonuclease activity, which serves as proofreading

– If pol I added wrong nt, won’t base pair properly– Pol I pauses, exonuclease removes mispaired nt– Allows replication to continue– Increases fidelity of replication

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Klenow Fragment Structure

• Wide cleft for binding to DNA between two -helices like a hand– One helix is part of the “fingers”– Other helix serves as the “thumb” domain– Between the helices lies a -sheet, palm

• 3 conserved Asp residues• Essential for catalysis• Likely coordinate Mg2+ (metal ions)

• Polymerase activity is separated from the exonuclease activity

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5’3’ exonuclease

• This activity allows pol I to degrade a strand ahead of advancing polymerase

• Removes and replaces a strand in one pass

• Basic functions are:– Primer removal– Nick repair

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Polymerases II and III

• Pol II activity is not required for DNA replication

• Pol I appears mostly active in repair

• Only Pol III is required for DNA replication– Pol III is the enzyme that replicates bacterial

DNA

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The Pol III Holoenzyme

• Pol III core is composed of 3 subunits:– DNA polymerase activity is in the -subunit– 3’5’exonuclease activity found in -subunit– Not yet clear what is the role of -subunit

• DNA-dependent ATPase activity is located in the -complex containing 5 subunits

• Lastly, -subunit plus the other 8 comprise the holoenzyme

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Fidelity of Replication

• Faithful replication is essential to life• DNA replication machinery has a built-in

proofreading system– This system requires priming– Only a base-paired nucleotide can serve as a

primer for Pol III holoenzyme– If wrong nucleotide is incorporated

accidentally replication stalls until 3’5’ exonuclease of Pol III holoenzyme removes it

• Primers are made of RNA which may help mark them for degradation

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Multiple Eukaryotic DNA Polymerases

Mammalian cells contain at least 5 different DNA polymerases

– Polymerases and appear to participate in replicating both DNA strands

– Priming DNA synthesis is -subunit role– Elongating both strands is done by -subunit

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Strand Separation

• DNA replication assumes that the 2 DNA strands at the fork somehow unwind

• This does not happen automatically as DNA polymerase does its job– 2 parental strands hold tightly to each other– This takes energy and enzyme action to

separate them– Helicase that unwinds dsDNA at the

replicating fork is encoded by E. coli dnaB gene

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Single-Strand DNA-Binding Proteins

• Prokaryotic ssDNA-binding proteins bind much more strongly to ssDNA than to dsDNA– Aid helicase action by binding tightly and

cooperatively to newly formed ssDNA– Keep it from annealing with its partner

• By coating ssDNA, SSBs protect it from degradation

• SSBs are essential for prokaryotic DNA replication

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Topoisomerases

• Strand separation of DNA is referred to as “unzipping”– DNA is not really like a zipper with straight, parallel

sides, actually a double helix– When 2 strands of DNA separate, rotate around each

other

• Helicase could handle this task alone if DNA were linear, short

• Closed circular DNA present special problems– As DNA unwinds at one site– More winding must occur at another site

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Cairns’s Swivel Concept

• A “swivel” in the DNA duplex called DNA gyrase

• Allows the DNA strands on either side to rotate to relieve the strain

• Gyrase belongs to the enzyme class topoisomerase

• These add transient single- or double-stranded breaks into DNA

• Serves to permit change in shape or topology

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Topoisomerase Mechanism

• Enzymes called helicases use ATP energy to separate the two parental DNA strands at the replication fork

• As helicase unwinds 2 parental strands it introduces a compensating positive supercoiling force

• Stress of this force must be overcome or DNA will resist progression of replication fork

• This stress releasing mechanism is the swivel• DNA gyrase acts as swivel by pumping negative

supercoils into replicating DNA

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20.3 DNA Damage and Repair

• DNA can be damaged in many different ways, if left unrepaired this damage can lead to mutation, changes in the base sequence of DNA

• DNA damage is not the same as mutation though it can lead to mutation

• If a particular kind of DNA damage is likely to lead to a mutation, we call it genotoxic

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Definition of DNA Damage

• DNA damage is a chemical alteration– Mutation is a change in a base pair

– Common examples of DNA damage• Base modifications caused by alkylating agents

• Pyrimidine dimers caused by UV radiation

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Damage Caused by Alkylation of Bases

• Alkylation is a process where electrophiles:– Encounter negative

centers– Attack them– Add carbon-containing

groups (alkyl groups)

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Damage Caused by Alkylation of BasesAlkylating agents like ethylmethane sulfonate (EMS) add alkyl groups to bases

– Some alkylation don’t change base-pairing, innocuous– Others cause DNA replication to stall

• Cytotoxic• Lead to mutations if cell attempts to replicate without

damage repair– Third type change base-pairing properties of a base, so are

mutagenic

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Damage Caused by Radiation

• Ultraviolet rays – Comparatively low energy– Result in formation of pyrimidine dimers, also

called cyclobutane pyrimidine dimers (CPDs)

• Gamma and x-rays– Much more energetic– Ionize molecules around the DNA– Form highly reactive free radicals that attack

DNA• Alter bases• Break strands

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DNA Damage: Pyrimidine Dimers and 8-oxoguanine

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Directly Undoing UV DNA Damage

• UV radiation damage to DNA can be directly repaired by a photolyase, which is actually two separate enzymes that catalyze repair of CPDs

• Uses energy from near-UV to blue light to break bonds holding 2 pyrimidines together

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Undoing High Energy DNA Damage

• O6 alkylations on guanine residues can be directly reversed by the “suicide enzyme”, O6-methylguanine methyltransferase

• This enzyme accepts the alkyl group onto the sulfur group of one of its cysteines and becomes irreversibly inactivated

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Excision Repair

• Percentage of DNA damage products that can be handled by direct reversal is small

• Most damage involves neither pyrimidine dimers nor O6-alkylguanine

• Another repair mechanism is required, excision repair is the process that removes most damaged nucleotides– Damaged DNA is removed– Replaced with fresh DNA– Base and nucleotide excision repair are both

used, BER and NER, respectively

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Base Excision Repair

Base excision repair (BER) acts on subtle base damage

– Begins with DNA glycosylase• Extrudes a base in a damaged base pair• Clips out the damaged base• Leaves an apurinic or apyrimidinic site that attracts

DNA repair enzymes

– DNA repair enzymes • Remove the remaining deoxyribose phosphate• Replace it with a normal nucleotide

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Base Excision Repair in E. coli

• DNA polymerase I fills in missing nucleotide in BER

• Base is removed the AP site remains – apurinic or apyrimidinic

• AP endonuclease cuts or nicks DNA strand

• Phosphodiesterase removes the AP sugar phosphate

• Pol I performs repair synthesis

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Eukaryotic BER

• DNA polymerase fills in the missing nucleotide– Makes mistakes– No proofreading activity

• APE1 carries out proofreading• Repair of 8-oxyguanine (oxoG) sites in DNA is

special case BER – can occur in 2 ways– A that has mispaired with oxoG can be removed after

DNA replication by a specialized adenine DNA glycosylase

– oxoG will still be paired with C and oxoG removed by another DNA glycoslyase, oxoG repair enzyme

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Nucleotide Excision Repair

• Nucleotide excision repair typically handles bulky damage that distorts DNA double helix

• NER in E. coli begins when damaged DNA is clipped by an endonuclease on either side of the lesion, sites 12-13 nt apart

• Allows damaged DNA to be removed as part of resulting 12-13-base oligonucleotide

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NER in E. coli

• Excinuclease (UvrABC) cuts either side• Remove oligonucleotide 12-13 nt• DNA polymerase I fills in missing nucleotides

using top strand as template• DNA ligase seals the nick to complete the task

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Eukaryotic NER

• Eukaryotic NER uses 2 paths• GG-NER (global genome)

– Complex composed of XPC and hHR23B initiates repair binding lesion in the genome

– Causes limited amount of DNA melting– XPA and RPA are recruited– TFIIH joins, 2 subunits (XPB, XPD) use

helicase to expand the melted region– RPA binds 2 excinucleases (XPF, XPG)

positions for cleavage– Releases damaged fragment 24-32 nt long

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Congenital defects in DNA Repair

• Much of our information about repair mechanisms in humans has come from the study of congenital defects in DNA repair

• These repair disorders cause a group of human diseases, including Cockayne’s syndrome and xeroderma pigmentosum (XP)

• Most XP patients are thousands of times more likely to develop skin cancer when exposed to the sun compared to healthy persons without XP

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Transcription-Coupled NER

• TC-NER is very similar to GG-NER except:– RNA polymerase plays role of XPC in damage

sensing and initial DNA melting

• In either type, DNA polymerase or fills in the gap left by removal of damaged fragment

• DNA ligase seals the DNA

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Human Global Genome NER

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Double-Strand Break Repair in Eukaryotes

• dsDNA breaks in eukaryotes are probably most dangerous form of DNA damage

• These are really broken chromosomes– If not repaired lead to cell death– In vertebrates can also lead to cancer

• Eukaryotes deal with dsDNA breaks in 2 ways:– Homologous recombination– Nonhomologous end-joining (NHEJ)

• Role of chromatin remodeling in dsDNA break repair

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Model for Nonhomologous End-Joining• This process requires Ku and

DNA-PKcs which bind at DNA ends and lets ends find regions of microhomology

• 2 DNA-PK complexes phosphorylate each other and activates– Catalytic subunit to dissociate– DNA helicase activity of Ku to

unwind DNA ends

• Extra flaps of DNA removed, gaps filled, ends permanently ligated

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Role of Chromatin Remodeling in Double-Stranded Break Repair

• 2 protein kinases, Mec1 and Tel1, are recruited to DSBs

• They phosphorylate Ser129 of histone H2A in nearby nucleosomes

• Phosphorylation recruits chromatin remodeler IN080 to the DSB– Use DNA helicase activity to push nucleosomes away

from DSB ends– Forms ssDNA overhangs essential for recombination

• SWR1 shares components with IN080– Replaces phosphorylated H2A with variant Htz1

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Mismatch Repair

• Mismatch repair system recognizes parental strand by methylated A in GATC sequence

• Corrects mismatch in progeny strand

• Eukaryotes use part of repair system

• Rely on different, uncharacterized method to distinguish strands at a mismatch

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Coping with DNA Damage Without Repairing It

• Direct reversal and excision repair are true repair processes

• Eliminate defective DNA entirely

• Cells can cope with damage by moving around it– Not true repair mechanism– Better described as damage bypass

mechanism

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Recombination Repair

• The gapped DNA strand across from a damaged strand recombines with normal strand in the other daughter DNA duplex after replication

• Solves gap problem• Leaves original damage

unrepaired

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Error-Prone Bypass

• Induce the SOS response

• This causes DNA to replicate even though the damaged region cannot be read correctly

• Result is errors in the newly made DNA

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Error-Free Bypass in Humans

• Humans have relatively error-free bypass system that inserts dAMPs across from pyrimidine dimers

• Replicate thymine dimers correctly

• Uses DNA polymerase plus another enzyme to replicate a few bases beyond the lesion

• If DNA polymerase gene is defective, DNA polymerase and others take over

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Error-Prone Bypass in Humans

• Errors in correcting UV damage lead to a variant form of XP, XP-V

• DNA polymerase is active on templates with thymidine dimers and AP sites

• The polymerase is not error-free

• With a gapped template, it is one of the least accurate template-dependent polymerases known