Molecular biology - Fourth edition - Chapter 21: DNA replication II: Detailed mechanism

Molecular BiologyFourth EditionChapter 21DNA Replication II:Detailed MechanismLecture PowerPoint to accompanyRobert F. WeaverCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.121.1 InitiationInitiation of DNA replication means primer synthesisDifferent organisms use different mechanisms to make primersDifferent phages infect E. coli using quite different primer synthesis strategiesColiphages were convenient tools to probe DNA replication as they are so simple they must rely primarily on host proteins to replicate their DNAs2Priming in E. coliPrimosome refers to collection of proteins needed to make primers for a given replicating DNAPrimer synthesis in E. coli requires a primosome composed:DNA helicaseDnaBPrimase, DnaGPrimosome assembly at the origin of replication, oriC uses multi-step sequence3Priming at oriCSource: Adapted from DNA Replication, 2/e, (plate 15) by Arthur Kornberg and Tania Baker.4Origin of Replication in E. coliPrimosome assembly at oriC occurs as follows:DnaA binds to oriC at sites called dnaA boxes and cooperates with RNA polymerase and HU protein in melting a DNA region adjacent to leftmost dnaA boxDnaB binds to the open complex and facilitates binding of primase to complete the primosomePrimosome remains with replisome, repeatedly primes Okazaki fragment synthesis on lagging strandDnaB has a helicase activity that unwinds DNA as the replisome progresses5Priming in EukaryotesEukaryotic replication is more complex than bacterial replicationComplicating factorsBigger size of eukaryotic genomesSlower movement of replicating forksEach chromosome must have multiple originsStarted study with a simple monkey virusLater consider yeast6Origin of Replication in SV40The SV40 origin of replication is adjacent to the viral transcription control regionInitiation of replication depends on the viral large T antigen binding to: Region within the 64-bp ori coreTwo adjacent sitesExercises a helicase activity that opens up a replication bubble within the ori corePriming is carried out by a primase associated with host DNA polymerase a7Origin of Replication in YeastYeast origins of replication are contained within autonomously replicating sequences (ARSs)These are composed of 4 important regions:Region A is 15 bp long and contains an 11-bp consensus sequence highly conserved in ARSsB1 and B2B3 may allow for an important DNA bend within ARS1821.2 ElongationOnce a primer is in place, real DNA synthesis can beginAn elegant method of coordinating the synthesis of lagging and leading strands keep the pol III holoenzyme engaged with the templateReplication can be highly processive and so very rapid9Speed of ReplicationThe pol III holoenzyme synthesizes DNA at the rate of about 730 nt/sec in vitroThe rate in vivo is almost 1000 nt/secThis enzyme is highly processive both in vitro and in vivo10The Pol III Holoenzyme and Processivity of ReplicationPol III core alone is a very poor polymerase, after assembling 10 nt it falls off the templateTakes about 1 minute to reassociate with the template and nascent DNA strandSomething is missing from the core enzymeThe agent that confers processivity on holoenzyme allows it to remain engaged with the templateProcessivity agent is a “sliding clamp”, the b-subunit of the holoenzyme11The Role of the b-SubunitCore plus the b-subunit can replicate DNA processively at about 1,000 nt/secDimer formed by b-subunit is ring-shapedRing fits around DNA templateInteracts with a-subunit of the core to tether the whole polymerase and template togetherHoloenzyme stays on its template with the b-clampEukaryotic processivity factor, PCNA forms a trimer, also forms a ring that encircles DNA and holds DNA polymerase on the template12The Clamp LoaderThe b-subunit needs help from the g complex to load onto the DNA templateThis g complex acts catalytically in forming this processive adb complexDoes not remain associated with the complex during processive replicationClamp loading is an ATP-dependent processEnergy from ATP changes conformation of the loader so that d-subunit binds to one of the b-subunits of the clampThis binding opens the clamp and allows it to encircle DNA13The b Clamp and LoaderSource: Adapted from Ellison, V. and B. Stillman, Opening of the clamp: An intimate view of an ATP-driven biological machine. Cell 106(2001), p. 657, f. 3.Source: Adapted from Henderson, D.R. and T.J. Kelly, DNA polymerase III: Running rings around the fork. Cell 84:6, 1996.14Lagging Strand SynthesisThe pol III holoenzyme is double-headedThere are 2 core polymerases attached through 2 t-subunits to a g complexOne core is responsible for continuous synthesis of the leading strandOther core performs discontinuous synthesis of the lagging strandThe g complex serves as a clamp loader to load the b clamp onto a primed DNA templateAfter loading, b clamp loses affinity for g complex instead associating with core polymerase15Simultaneous Strand SynthesisThe g complex and b clamp help core polymerase with processive synthesis of an Okazaki fragmentWhen fragment completed, b clamp loses affinity for coreAssociate b clamp with g complex which acts to unload clampNow clamp recycles16Lagging Strand ReplicationSource: Adapted from Henderson, D.R. and T.J. Kelly, DNA polymerase III: Running rings around the fork. Cell 84:7, 1996.1721.3 TerminationTermination of replication is straightforward for phage that produce long, linear concatemersConcatemer grows until genome-sized piece is snipped off and packaged into phage headBacterial replication – 2 replication forks approach each other at the terminus regionContains 22-bp terminator sites that bind specific proteins (terminus utilization substance, TUS)Replicating forks enter terminus region and pauseLeaves 2 daughter duplexes entangledMust separate or no cell division18Decatenation: Disentangling Daughter DNAsAt the end of replication, circular bacterial chromosomes form catenanes that decatenated in a two step processFirst, remaining unreplicated double-helical turns linking the two strands are meltedRepair synthesis fills in the gapsLeft with a right-handed parallel torus catenane with an even number nodes that is decatenated by topoisomerase IVLinear eukaryotic chromosomes also require decatenation during DNA replication19Termination in EukarytoesUnlike bacteria, eukaryotes have a problem filling the gaps left when RNA primers are removed at the end of DNA replicationIf primer on each strand is removed, there is no way to fill in the gapsDNA cannot be extended 3’5’ directionNo 3’-end is upstreamIf no resolution, DNA strands would get shorter with each replication20Telomere MaintenanceAt the ends of eukaryotic chromosomes are special structures called telomeresOne strand of telomeres is composed of tandem repeats of short, G-rich regions whose sequence varies from one species to anotherG-rich telomere strand is made by enzyme telomeraseTelomerase contains a short RNA serving as template for telomere synthesisC-rich telomere strand is synthesized by ordinary RNA-primed DNA synthesisThis process is like lagging strand DNA replicationThis mechanism ensures that chromosome ends can be rebuilt and do not suffer shortening with each round of replication21Telomere Formation22Telomere StructureAll eukaryotes protect their telomeres from nucleases and ds break repair enzymesCiliates have TEBP to bind and protect the 3’-single-strand telomeric overhangBudding yeast has Cdc13p which recruits Stn1p and Ten1p that all bind ss telomeric DNAMammals and fission yeast also have a protein similar to TEBP binding specifically to ss telomeric DNA23Mammalian Telomere StructureMammalian telomeres form looped structures that protect ss telomeric DNAProteins TRF1 and TRF2 appear to help telomeric DNA in mammalian cells form a t loop in which ss 3’-end of telomere invades the ds telomeric DNA upstreamTRF1 may help bend the DNA into shape for strand invasionTRF2 binds at point of strand invasion, may stabilize the displacement loop24