REPLICATION

REPLICATIONThe ProblemModels of ReplicationSlide 4Slide 5Slide 6Slide 7Extending the ChainSlide 9DNA SynthesisSlide 11Semi-discontinuous ReplicationOkazaki ExperimentSlide 14Features of DNA ReplicationDNA Replication-ProkaryotesThe Problem of OverwindingTopoisomerase Type IHelicaseGyrase--A Type II TopoisomeraseInitiation of ReplicationDe novo InitiationUnwinding the DNA by Helicase (DnaB protein)Liebowitz ExperimentLiebowitz Assay--ResultsSingle Stranded DNA Binding Proteins (SSB)Replication: The OverviewThe DNA Polymerase FamilySlide 29DNA pol ISlide 31Slide 32Nick TranslationNick Translation 2DNA Polymerase I is great, but….Other clues….DNA Polymerase IIIDNA Polymerase III Holoenzyme (Replicase)Activities of DNA Pol IIISlide 40Leading and Lagging StrandsThe ReplisomeLigaseDNA Replication ModelTermination of ReplicationFIDELITY OF REPLICATIONWhy Okazaki Frags?REPLICATIONREPLICATIONChapter 7Chapter 7The 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 unwoundTHREE HYPOTHESES FOR DNA REPLICATIONModels of ReplicationModels of Replication(a) Hypothesis 1:Semi-conservative replication(b) Hypothesis 2:Conservative replicationIntermediate molecule(c) Hypothesis 3:Dispersive replicationMODELS OF DNA REPLICATIONPREDICTED DENSITIES OFNEWLY REPLICATED DNAMOLECULES ACCORDINGTO THE THREE HYPOTHESESABOUT DNA REPLICATIONMeselson and Stahl Conclusion: Semi-conservative replication of DNAReplication 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 ChainDNA SynthesisDNA Synthesis2 phosphates3’-OH nucleophilic attack on alpha phosphate of incoming dNTPremoval and splitting of pyrophosphate by inorganic pyrophosphataseChain Elongation in the 5’  3’ directionSemi-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 fragmentsOkazaki ExperimentOkazaki ExperimentContinuous synthesisDiscontinuous synthesisDNA replication is semi-discontinuousFeatures 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 directionsdirectionsDNA Replication-Prokaryotes DNA Replication-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: TopoisomerasesThe Problem of OverwindingThe Problem of OverwindingTopoisomerase 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 ATPGyrase--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 bidirectionallbidirectionallyyUnwinding the DNA by Helicase Unwinding the DNA by Helicase (DnaB protein)(DnaB protein)Uses ATP to separate