MCB 502A-2014. Lecture#11. The Cell Cycle.— The prokaryotic versus eukaryotic cell cycle— Helmstetter's "baby machine"— Replication period as a function of growth rate— The regulation of chromosomal initiation: titration of DnaA— The initiation cascade reflects the eclipse period— Flow cytometry reveals initiation synchrony— Dam and the origin sequestration— SeqA: the eclipse and beyond— Nucleoid compaction and administration— The chromosome cycleThe prokaryotic cell cycleThe prokaryotic versuseukaryotic cell cycle-1— Chromosomal replication is the central eventof the cell cycle, which is the sequence ofevents that initiates with the birth of a new celland culminates with its division to give rise totwo daughter cells.— The familiar outline of the eukaryotic cellcycle is represented by the circular diagram—>G1—>S—>G2—>M—>.G2SD(M)G1(replication)(segregation)2n1nGenomecontentper cellG1 S G2 D(M)— Another way to presentthe cell cycle is via thegenome content of the cell: itis stable in Gap-1, duplicatesthroughout S, stable again inGap-2 and is halved by celldivision.The prokaryotic versuseukaryotic cell cycle-2— We know that the eukaryotic cell cycleis driven by the cycline-dependent kinases(CDKs) and is subdivided into four majorblocks of activities.— When CDK activates a particular blockof activities, it then waits for theinformation about completion of theseactivities.— Once all the activities of the block havebeen successfully completed, CDKactivates the next block of the cell cycle,and so on.— The critical feature of the eukaryoticcell cycle is that any particular step cannotbegin before the previous step has beenreported as accomplished.G2SD(M)G1CDKThe prokaryotic versuseukaryotic cell cycle-3— Can we study the bacterial cell cycle thesame way we study the eukaryotic one?— In particular, can we detect the period ofDNA synthesis by following incorporation of aradiolabeled DNA precursors into DNA?— If we just add label to a growing culture andmeasure the rate of label incorporation intoDNA, we will end up with an exponentialcurve, which would be superimposable with thegrowth curve of the culture.— This result is uninterpretable (and would besimilarly uninterpretable in eukaryotic systems),because the culture is asynchronous: some cellshave just started chromosomal replication, someare finishing it while still others are engaged inchromosomal segregation and cell division.% TCA-precipitable 3H-counts3H-dTTimeGrowthThe prokaryotic versuseukaryotic cell cycle-4— Synchronization is a prerequisite forstudying the cell cycle in any system.— As was discussed at the last lecture, wecan synchronize the culture if we preventreplication initiation by blocking proteinsynthesis due to withdrawal of a requiredaminoacid,(1) waiting until all cells complete theongoing rounds of DNA replication andthen(2) releasing the protein synthesis block, sothat replication initiation occurs at the sametime in all chromosomes of the population.12The prokaryotic versuseukaryotic cell cycle-5— In slow-growingsynchronized cells of E. coli,we see the familiar features ofthe eukaryotic cell cycle.— After the cell division,there is a period of no DNAsynthesis (Gap1), followed bya period during which DNA issynthesized (S), followed by ashort lull in activities (Gap2),followed by cell division (D).— Essentially the same curvecan be obtained for HeLa(~human) cultured cells.2n1nGenomecontentper cellG1 S G2 D(M)http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2973560/http://www.sciencedirect.com/science/article/pii/0014482763903069#The prokaryotic versuseukaryotic cell cycle-6— What is the mechanism driving this bacterialcell cycle?— Since it looks superficially similar to theeukaryotic one, we may start with a simple ideathat the bacterial cell cycle is driven by ananalog of cycline-dependent kinase, with asimilar format of orders to execute specificstages and decisions made on the basis ofreports of successful completion of prior stages.— This system assumes the existence of agroup of dedicated signal-generating andinformation-processing proteins (kinases) thatdo nothing else besides driving the cell cycle,and whose inactivation is lethal (like CDKs ineukaryotic cells).— Kind of cells' CEOs.G2SD(M)G1CDKThe prokaryotic versuseukaryotic cell cycle-7— No such system has been revealed so farin bacteria; in particular, there is no groupof conditionally-lethal mutants in suspectedprotein kinases.— The majority of lethal mutations in E.coli directly inactivate important metabolicfunctions, like DNA ligase or the initiationprotein DnaA, that execute, rather thanorder, a particular stage of the cell cycle.eukaryotesprokaryotesThe prokaryoticversus eukaryoticcell cycle-8— Perhaps we can get a glimpse into thebacterial cell cycle if we study bacteriagrowing at different division rates?— We know that in the lab, in the mostfavorable growth conditions, entericbacteria like E. coli can divide as fast asevery 24 minutes.— In natural habitats, enteric bacteria growmuch slower.— E. coli is distributed 50:50 between thetwo natural habitats: the guts of vertebrates,where bacterium is thought to live a stablethough very slow life, and natural waters oflakes and ponds, where bacterium isstarved and dying.The prokaryoticversuseukaryotic cellcycle-9— When in the water, the bacteriumdoes not multiply and simply tries tostay alive, waiting to be swallowed byan animal with an appropriate gut.— Once in an appropriate gut, withplenty of food around, a luckybacterium will try to colonize it.— However, there is a lot ofcompetition in established gutcommunities from the resident species.— As a result, bacteria in the gut arethought to divide only once a day, —judging by the total mass of bacteriathere and by the portion of this massthat gets "evacuated" on the regularbasis.The prokaryotic versuseukaryotic cell cycle-10— Thus, under the most optimal conditionsin the lab, E. coli divides once every 24minutes, whereas during the stable growthin the gut it divides once every 24 hours,with a 60 times longer cell cycle.— We may further presume that thechromosomal replication probably takesless than 20 minutes when cells aredividing every 24 minutes, or it couldeasily take 20 hours when cells are dividingevery 24 hours.— The way to test this notion would be tomeasure the timing and length ofchromosomal replication in
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