MCB 502A-2013. Lecture #10.Initiation and Termination.— Replication factoryInitiation of the chromosomal replication in E. coli— Origin of replication, oriC— DnaA and DnaC— oriC-dnaA-independent initiationTermination of the chromosomal replication— Discovery of a termination site— Tus protein and the replication fork trap— Topo IV: decatenation of the sister chromatidsThe prokaryotic cell cycle— The prokaryotic versus eukaryotic cell cycleLast time: the static replisomeα + ε + θα + ε + θα + ε + θThe StaticReplisomeReplication factory-1— Since replication forks always come inpairs, each defining the opposite sides of areplication bubble, and both replisomes arestatic, there is no sense in keeping themseparate!— It was argued that the two replisomes of thebubble are combined in a single hyperstructure,into which two parental DNA duplexes enterand from which four daughter duplexes exit.— Such a protein complex in combination witha replication DNA bubble is called the"replication factory": the place where all thereplication enzymes are organized together,creating optimal conditions for their functionand to where all the DNA that needs to bereplicated has to come.Static ReplisomesReplication factory-2— The idea of the replicationfactory combining two tworeplisomes serving the samereplication bubble wasproposed by Wesley Dingmanback in 1974…Replication factory-3— … but was tested only in 1998 by KatherineLemon and Alan Grossman, by fusing thereplicative DNA polymerase in Bacillus subtiliswith green fluorescent protein (GFP).— In these cells, the green fluorescent dots markedthe position of the replication complex.http://proteopedia.org/wiki/index.php/Green_Fluorescent_ProteinReplication factory-4— If the chromosome is static, while tworeplisomes of the bubble are moving along it,there should be no specific place in the cell forthe replisomes, whereas the number of GFPspots should be equal to the number ofreplication forks.— If, however, the replication factory is static,while DNA is drawn through it, then there willbe a fixed position for the factory in the cell,and there should be one GFP spot per tworeplication forks.Replication factory-5— Lemon and Grossman found the latterdistribution in B. subtilis.— Specifically, in slowly-growing cells with asingle replication bubble per chromosome,there was either a single GFP focus per cell atthe position 1/2 of the cell length, or (muchless frequently) two foci, representing twosegregated and replicating nucleoids.— In rapidly-growing cells, that have threereplication bubbles, there were three GFP foci:at 1/4, 1/2 and 3/4 of the cell length.— Not only the number of foci matched thenumber of replication bubbles, rather thanindividual forks, but the position of these fociin the cell was more-or-less fixed, as predictedby the replication factory model.# of cellsCell length (%)100# of cellsCell length (%)100100# of cellsCell length (%)Replication factory-6— Another strong prediction of thereplication factory model was that anyDNA associates with the replicationfactory only transiently.— To test this, in addition to the PolC-GFP labeling (with green color) therewas replication origin labeling added,with a red color (this is a bit ahead of thenext material).— In the aligned cultures (also a bitahead), the origin label coincided withthe replication factory label, as expected.— However, in the cells that startedDNA replication, the origin label movedto two separate positions, away from thestill central replication factory.Aligned cultureReleased cultureReplicationfactory-6— This experimentdemonstrated not onlythat DNA sequencescome to the replicationfactory to replicate, ……but also that, oncereplicated, they leave thefactory and assume theirdistinct positions withinthe cell.Mother nucleoidDaughter nucleoid Daughter nucleoid"Replication factory"Sister-chromatidcohesion stationReplication factory-7— In contrast to B. subtilis, where areplication bubble is served by a singlereplication factory, the same experiment inE. coli cells with a single replication bubbleyielded mostly two foci that movedindependently of each other, but withinnarrow confines around positions 1/3 and2/3 of the cell length.— Thus, the idea of a single replicationfactory may not be applicable broadly,although it is clear that even if the tworeplisomes of the same bubble are separatein slow-growing E. coli, they are stillstationary and do not move around thechromosome, as was imagined before.# of cellsCell length (%)Replication factory-8— Interestingly, in fast-growing E. coli cellswith three replication bubbles, the individualreplication forks do come together, perhaps evenforming replication factories.— Even more importantly, the number ofreplication foci in the nuclei of higher eukaryoticcells matches the expected number of replicationbubbles, rather than the 2x-higher number ofindividual replication forks, indicatingreplication factories in these cells as well.# of cellsCell length (%)EdU labeling of nascent DNA Anti-PCNA antibody MERGEhttp://openi.nlm.nih.gov/detailedresult.php?img=2803164_1471-2121-10-88-1&req=4Check out howour replicationideas havedeveloped!— Our replication conceptskeep their steady development,from the template strandsurrounded by DNA precursorsof the mid-1950s to this DNAreplication at the central factorywith subsequent segregation ofthe two daughters away fromthe factory and in the oppositedirections.5'Initiation of the chromosomalreplication in E. coliOrigin of replication, oriC-1— Recall the seminal demonstration ofCairns of a single replication bubble in E.coli, corroborated by the laterdemonstration by Prescott and Kuempelthat both replication forks of the bubbleare active.— The next question in that line ofresearch was about the location of thereplication origin: is it fixed or is itrandom?— This could have been done in asynchronized culture (where all the cellsbegin chromosomal replication at thesame time) if one can determine thetiming of duplication of various pointsalong the chromosome.Replication forks!Origin of replication, oriC-2— Caro decided to address this question with a set of strains each harboring asingle prophage Mu inserted at various (mapped) locations around thechromosome.— The Mu prophage, that inserts randomly, served as a variously-positionedsegment of homology, to which a radioactive probe (labeled Mu DNA) could behybridized.— Another
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