Bài giảng Molecular Biology - Chapter 20 DNA Replication, Damage, and Repair

Molecular BiologyFifth EditionChapter 20DNA Replication, Damage, and RepairLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.120.1 General Features of DNA ReplicationDouble helical model for DNA includes the concept that 2 strands are complementaryEach strand can serve as template for making its own partnerSemiconservative model for DNA replication is correctHalf-discontinuous (short pieces later stitched together)Requires DNA primersUsually bidirectional2Three Hypotheses of ReplicationThe three methods of DNA replication considered were:Semiconservative Conservative Dispersive 3DNA replicates in a semiconservative mannerWhen parental strands separateEach strand serves as templateMakes a new, complementary strandSemiconservative Replication4Semidiscontinuous ReplicationDNA replication in E. coli (and in other organisms) is semidiscontinuousOne strand (the leading strand) is replicated continuously in the direction of the movement of the replicating forkThe other strand (the lagging strand) is replicated discontinuously as 1-2 kb Okazaki fragments in the opposite directionThis allows both strands to be replicated in the 5’3’-direction5DNA Replication Models6Priming DNA SynthesisOkazaki fragments in E. coli are initiated with RNA primers 10-12 nt longIntact primers are difficult to detect in wild-type cells because of enzymes that attack RNAs7Bidirectional ReplicationThe 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 synthesizedAs the bubble expands, replicating DNA begins to take on the  shape8Theta Mode of DNA Replication in E.coli9Replication ForkIn DNA replication, the replication forks represent the sites of DNA replicationDirection 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 originOrigin of replication is the fixed starting point for DNA replicationThe replicon is the DNA under the control of one origin of replication 10Rolling Circle ReplicationCircular DNAs can replicate by a rolling circle mechanismOne strand of a dsDNA is nicked and the 3’-end is extendedThis uses the intact DNA strand as a templateThe 5’-end is displacedPhage X174 replication cycles so that when one round is complete a full-length, single-stranded circle of DNA is releasedPhage l, displaced strand serves as the template for discontinuous, lagging strand synthesis11Phage l Rolling Circle ModelAs the circle rolls rightLeading strand elongates continuouslyLagging strand elongates discontinuouslyUses unrolled leading strand as a template RNA primers for Okazaki fragmentsProgeny dsDNA produced grows to many genomes before one genome worth is clipped off 1220.2 Enzymology of DNA ReplicationOver 30 different proteins or enzymes cooperate in replicating the E. coli DNAExamine the activities of some of these proteins and their homologs in other organismsStart with DNA polymerases – the enzymes that make DNA13E. coli DNA PolymerasesThere are 3 DNA polymerases, the enzymes that make DNA, found in E. coli: pol Ipol IIpol IIIE. coli DNA polymerase I was the first polymerase identifiedIt was discovered in 1958 by Arthur Kornberg14DNA Polymerase IDNA polymerase I (pol I) is a versatile enzyme with 3 distinct activitiesDNA polymerase3’5’ exonuclease5’3’ exonucleaseMild proteolytic treatment results in 2 polypeptidesKlenow fragment (the large domain)Smaller fragment15Klenow FragmentContains both: Polymerase and 3’5’ exonuclease activity, which serves as proofreadingIf pol I added wrong nt, won’t base pair properlyPol I pauses, exonuclease removes mispaired ntAllows replication to continueIncreases fidelity of replication16Klenow Fragment StructureWide cleft for binding to DNA between two -helices like a handOne helix is part of the “fingers”Other helix serves as the “thumb” domainBetween the helices lies a -sheet, palm3 conserved Asp residuesEssential for catalysisLikely coordinate Mg2+ (metal ions)Polymerase activity is separated from the exonuclease activity175’3’ exonucleaseThis activity allows pol I to degrade a strand ahead of advancing polymeraseRemoves and replaces a strand in one passBasic functions are:Primer removalNick repair18Polymerases II and IIIPol II activity is not required for DNA replicationPol I appears mostly active in repairOnly Pol III is required for DNA replicationPol III is the enzyme that replicates bacterial DNA19The Pol III HoloenzymePol III core is composed of 3 subunits:DNA polymerase activity is in the -subunit3’5’exonuclease activity found in -subunitNot yet clear what is the role of -subunitDNA-dependent ATPase activity is located in the g-complex containing 5 subunitsLastly, b-subunit plus the other 8 comprise the holoenzyme20Fidelity of ReplicationFaithful replication is essential to lifeDNA replication machinery has a built-in proofreading systemThis system requires primingOnly a base-paired nucleotide can serve as a primer for Pol III holoenzymeIf wrong nucleotide is incorporated accidentally replication stalls until 3’5’ exonuclease of Pol III holoenzyme removes itPrimers are made of RNA which may help mark them for degradation21Multiple Eukaryotic DNA PolymerasesMammalian cells contain at least 5 different DNA polymerasesPolymerases d and a appear to participate in replicating both DNA strandsPriming DNA synthesis is a-subunit roleElongating both strands is done by d-subunit22Strand SeparationDNA replication assumes that the 2 DNA strands at the fork somehow unwindThis does not happen automatically as DNA polymerase does its job2 parental strands hold tightly to each otherThis takes energy and enzyme action to separate themHelicase that unwinds dsDNA at the replicating fork is encoded by E. coli dnaB gene23Single-Strand DNA-Binding ProteinsProkaryotic ssDNA-binding proteins bind much more strongly to ssDNA than to dsDNAAid helicase action by binding tightly and cooperatively to newly formed ssDNAKeep it from annealing with its partnerBy coating ssDNA, SSBs protect it from degradationSSBs are essential for prokaryotic DNA replication24TopoisomerasesStrand separation of DNA is referred to as “unzipping”DNA is not really like a zipper with straight, parallel sides, actually a double helixWhen 2 strands of DNA separate, rotate around each otherHelicase could handle this task alone if DNA were linear, shortClosed circular DNA present special problemsAs DNA unwinds at one siteMore winding must occur at another site25Cairns’s Swivel ConceptA “swivel” in the DNA duplex called DNA gyraseAllows the DNA strands on either side to rotate to relieve the strainGyrase belongs to the enzyme class topoisomerase These add transient single- or double-stranded breaks into DNAServes to permit change in shape or topology26Topoisomerase MechanismEnzymes called helicases use ATP energy to separate the two parental DNA strands at the replication forkAs helicase unwinds 2 parental strands it introduces a compensating positive supercoiling forceStress of this force must be overcome or DNA will resist progression of replication forkThis stress releasing mechanism is the swivelDNA gyrase acts as swivel by pumping negative supercoils into replicating DNA2720.3 DNA Damage and RepairDNA can be damaged in many different ways, if left unrepaired this damage can lead to mutation, changes in the base sequence of DNADNA damage is not the same as mutation though it can lead to mutationIf a particular kind of DNA damage is likely to lead to a mutation, we call it genotoxic28Definition of DNA DamageDNA damage is a chemical alterationMutation is a change in a base pairCommon examples of DNA damageBase modifications caused by alkylating agentsPyrimidine dimers caused by UV radiation29Damage Caused by Alkylation of BasesAlkylation is a process where electrophiles:Encounter negative centersAttack themAdd carbon-containing groups (alkyl groups)30Damage Caused by Alkylation of BasesAlkylating agents like ethylmethane sulfonate (EMS) add alkyl groups to basesSome alkylation don’t change base-pairing, innocuousOthers cause DNA replication to stallCytotoxicLead to mutations if cell attempts to replicate without damage repairThird type change base-pairing properties of a base, so are mutagenic31Damage Caused by RadiationUltraviolet rays Comparatively low energyResult in formation of pyrimidine dimers, also called cyclobutane pyrimidine dimers (CPDs)Gamma and x-raysMuch more energeticIonize molecules around the DNAForm highly reactive free radicals that attack DNAAlter basesBreak strands32DNA Damage: Pyrimidine Dimers and 8-oxoguanine33Directly Undoing UV DNA DamageUV radiation damage to DNA can be directly repaired by a photolyase, which is actually two separate enzymes that catalyze repair of CPDsUses energy from near-UV to blue light to break bonds holding 2 pyrimidines together34Undoing High Energy DNA DamageO6 alkylations on guanine residues can be directly reversed by the “suicide enzyme”, O6-methylguanine methyltransferaseThis enzyme accepts the alkyl group onto the sulfur group of one of its cysteines and becomes irreversibly inactivated35Excision RepairPercentage of DNA damage products that can be handled by direct reversal is smallMost damage involves neither pyrimidine dimers nor O6-alkylguanineAnother repair mechanism is required, excision repair is the process that removes most damaged nucleotidesDamaged DNA is removedReplaced with fresh DNABase and nucleotide excision repair are both used, BER and NER, respectively36Base Excision RepairBase excision repair (BER) acts on subtle base damageBegins with DNA glycosylaseExtrudes a base in a damaged base pairClips out the damaged baseLeaves an apurinic or apyrimidinic site that attracts DNA repair enzymesDNA repair enzymes Remove the remaining deoxyribose phosphateReplace it with a normal nucleotide37Base Excision Repair in E. coliDNA polymerase I fills in missing nucleotide in BERBase is removed the AP site remains – apurinic or apyrimidinicAP endonuclease cuts or nicks DNA strandPhosphodiesterase removes the AP sugar phosphatePol I performs repair synthesis38Eukaryotic BERDNA polymerase  fills in the missing nucleotideMakes mistakesNo proofreading activityAPE1 carries out proofreadingRepair of 8-oxyguanine (oxoG) sites in DNA is special case BER – can occur in 2 waysA that has mispaired with oxoG can be removed after DNA replication by a specialized adenine DNA glycosylaseoxoG will still be paired with C and oxoG removed by another DNA glycoslyase, oxoG repair enzyme39Nucleotide Excision RepairNucleotide excision repair typically handles bulky damage that distorts DNA double helixNER in E. coli begins when damaged DNA is clipped by an endonuclease on either side of the lesion, sites 12-13 nt apartAllows damaged DNA to be removed as part of resulting 12-13-base oligonucleotide40NER in E. coliExcinuclease (UvrABC) cuts either sideRemove oligonucleotide 12-13 ntDNA polymerase I fills in missing nucleotides using top strand as templateDNA ligase seals the nick to complete the task41Eukaryotic NEREukaryotic NER uses 2 pathsGG-NER (global genome)Complex composed of XPC and hHR23B initiates repair binding lesion in the genomeCauses limited amount of DNA meltingXPA and RPA are recruitedTFIIH joins, 2 subunits (XPB, XPD) use helicase to expand the melted regionRPA binds 2 excinucleases (XPF, XPG) positions for cleavageReleases damaged fragment 24-32 nt long42Congenital defects in DNA RepairMuch of our information about repair mechanisms in humans has come from the study of congenital defects in DNA repairThese 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 XP43Transcription-Coupled NERTC-NER is very similar to GG-NER except:RNA polymerase plays role of XPC in damage sensing and initial DNA meltingIn either type, DNA polymerase e or d fills in the gap left by removal of damaged fragmentDNA ligase seals the DNA44Human Global Genome NER45Double-Strand Break Repair in EukaryotesdsDNA breaks in eukaryotes are probably most dangerous form of DNA damageThese are really broken chromosomesIf not repaired lead to cell deathIn vertebrates can also lead to cancerEukaryotes deal with dsDNA breaks in 2 ways:Homologous recombinationNonhomologous end-joining (NHEJ)Role of chromatin remodeling in dsDNA break repair46Model for Nonhomologous End-JoiningThis process requires Ku and DNA-PKcs which bind at DNA ends and lets ends find regions of microhomology2 DNA-PK complexes phosphorylate each other and activatesCatalytic subunit to dissociateDNA helicase activity of Ku to unwind DNA endsExtra flaps of DNA removed, gaps filled, ends permanently ligated47Role of Chromatin Remodeling in Double-Stranded Break Repair2 protein kinases, Mec1 and Tel1, are recruited to DSBsThey phosphorylate Ser129 of histone H2A in nearby nucleosomesPhosphorylation recruits chromatin remodeler IN080 to the DSBUse DNA helicase activity to push nucleosomes away from DSB endsForms ssDNA overhangs essential for recombinationSWR1 shares components with IN080Replaces phosphorylated H2A with variant Htz148Mismatch RepairMismatch repair system recognizes parental strand by methylated A in GATC sequenceCorrects mismatch in progeny strandEukaryotes use part of repair systemRely on different, uncharacterized method to distinguish strands at a mismatch49Coping with DNA Damage Without Repairing ItDirect reversal and excision repair are true repair processesEliminate defective DNA entirelyCells can cope with damage by moving around itNot true repair mechanismBetter described as damage bypass mechanism50Recombination RepairThe gapped DNA strand across from a damaged strand recombines with normal strand in the other daughter DNA duplex after replicationSolves gap problemLeaves original damage unrepaired51Error-Prone BypassInduce the SOS responseThis causes DNA to replicate even though the damaged region cannot be read correctlyResult is errors in the newly made DNA52Error-Free Bypass in HumansHumans have relatively error-free bypass system that inserts dAMPs across from pyrimidine dimersReplicate thymine dimers correctlyUses DNA polymerase  plus another enzyme to replicate a few bases beyond the lesionIf DNA polymerase  gene is defective, DNA polymerase  and others take over 53Error-Prone Bypass in HumansErrors in correcting UV damage lead to a variant form of XP, XP-VDNA polymerase  is active on templates with thymidine dimers and AP sitesThe polymerase is not error-freeWith a gapped template, it is one of the least accurate template-dependent polymerases known54