Chapter 6DNA replication, repair and recombinationThis is essential to understand •cancer•genetic change•evolution•key techniques used for molecular biology/ biotechnology•current antiviral therapeuticsObjectives• Be able to explain how genetic information isreplicated and the role of the proteins involved• Understand the general mechanisms of DNA alterationand repairBe#able#to:• Explain)the)first)experiment)that)showed)that)DNA)replica8on)is)semi9conserva8ve• Draw)a)piece)of)DNA,)labeling)the)5’)and)3’)ends)of)each)strand• Draw)the)DNA)opened)up)for)replica8on,)with)the)RNA)primersaDached,)and)label)the)5‘)and)3’)ends)of)the)RNA)primers• Draw)the)new)leading)strand• Draw)the)next)RNA)primers,)labeling)the)5’)and)3’)ends• Draw)the)DNA)being)made)on)the)lagging)strand• Describe)how)the)RNA)primer)is)removed,)and)explain)how)ligase)sealsthe)gap• Name,)and)describe)the)func8on)of,)the)key)enzymes)in)replica8on• Describe)what)telomerase)does• Sketch)two)mechanisms)of)DNA)repair• Outline)what)recombina8on)does• Describe)what)a)transposon)is,)and)how)it)can)copy)itself• Sketch)the)molecular)mechanism)used)by)a)retrovirus• Describe)how)acyclovir)and)AZT)work06_02_DNA template.jpgDNA ReplicationEach DNA strand can serve as a template for thesynthesis of a new complementary strandA-T and G-C complementary basesP-HO--P-OHHow is DNA replicated?Meselson-Stahl ExperimentDNA replication is semiconservativeSemiconservative replicationTo begin replication, DNA strands must be pried apartAre replication origins more likely to be A-T rich or G-C rich?Visualization of ReplicationDNA synthesis is catalyzedby DNA polymeraseNucleotides are added onlyto the 3’ end of a DNAstrandDNA synthesis is 5’ to 3’If DNA is synthesized only in the 5’ to 3’ directionhow are both strands replicated?No free 3’ end to extendDiscontinuous replication allows “backstitching”Discontinuous replicationDNA polymerase can add anucleotide only to an existing3’ base-pairTo start a new strand weneed a primerThe primer is a short (10 bp)RNA and is synthesized byDNA primaseExtension of the RNA primerby DNA polymerasegenerates the Okazakifragment (~200 bp)DNA polymerase willextend the Okazakifragment until it runs intothe next RNA primerThe RNA primer will thenbe degraded by aribonuclease and a DNApolymerase called Repairpolymerase replaces theRNA with DNADNA Ligase joins thefragments (5’ P to 3’ OH)Ligase is a key enzymeused in molecular biologyE. coli DNA Human DNAAdd ligase, ATPMake hybrid genes, proteins, or organisms…..Multiple proteins act together at the replication forkCoordinatedreplicationProteins involved in replication: study guide• Initiator proteins: helix opening at replication origin• DNA Polymerase: polymerization (5’-3’) proofreading (3’-5’)• DNA Primase: synthesis of short RNA primer• Ribonuclease: degradation of RNA primer• Repair polymerase: replace RNA with DNA• Ligase: joining of DNA fragments• DNA Helicase: unzipping DNA helix prior to replication• Single-stranded DNA-binding protein: prevent reanealing• Sliding clamp: keeps DNA polymerase attached to templateand on lagging strand, releases when Okazaki fragment iscompletedTelomerase is needed to replicate chromosome endsTelomeres also help the cell distinguish true chromosome endsDNA Polymerase also hasan error-correcting activitycalled ProofreadingCatalyzed by a 3’ to 5’exonuclease activityError rate is about 1 errorin 107 base pairsDespite theproofreading ability ofDNA polymerase,errors still occurPermanent changes inDNA sequence aremutationsMutations can bedeleterious, neutral oreven beneficial (basisfor evolution). It is notalways easy todistinguish betweendeleterious andbeneficialSomatic cells canaccumulate mutations overa lifespan that may impacthealthDNA repair mechanismssafeguard DNA sequencesfrom random mutation06_21_Errors corrected.jpgDNA mismatch repairReplication error rate (DNA polymerase) is 1 in 107 basepairsDNA mismatch repair corrects 99% of these for an overall rate of 1 in 109DNA mismatch repairproteins recognize thenewly synthesized strandand selectively excise andresynthesize this strandRecognition probablyinvolves nicks in the newlysynthesized strandCommon chemical reactions knownto cause DNA damageDNA damage can lead to mutationsubstitution deletionGeneral mechanismof DNA repair1. Recognition and excision2. Replacement3. JoiningRecombinationRearrangement of DNA sequences can provide variationLike mutation, recombination can be either:• Neutral• Beneficial• DeleteriousTypes of recombination:• Homologous• Non-homologousHomologous recombinationRequires alignment of homologous sequences06_29_Homol.recomb2.jpgHomologous recombinationNo alteration of sequenceat the site of exchangeNonhomologous recombination(called site-specific recombination in the text)DNA exchange that is independent of sequenceNo homology is requiredUsually involves insertion or deletion of DNATransposons are common mobile genetic elementsTransposase is a recombination protein necessary fortransposon mobility. Recognizes transposon ends (red)Transposon movement mechanismsAmplification of transposonRetrotransposons move via an RNA intermediate L1 element (15% human genome)Alu element (11% human genome)Many transposons present in the humangenome are not capable of further movementLytic phaseLysogenic phaseNonhomologousrecombination is akey step in thelysogenic phase ofviral reproduction06_39_retrovirus.jpgRetroviruses foundonly in eukaryotic cellsGNo 3’ OH, sonext nucleotide can’tbe added to the chain -Stops herpesvirusDNA replicationAcyclovir -Frontline therapeutic forHerpesvirus - looks likea G base and the top ofthe ribose sugarNo 3’ OH, sonext nucleotide can’tbe added to the chain -Stops HIV DNAsynthesisAzidothymidine -Frontline therapeutic forHIV - looks like a Tbase and a ribose witha modified 3’ OHUseful text questions6-26-36-46-56-66-76-86-106-12, 13, 14, 15, 16,