MCB 502A-2015. Lecture #10. Initiation and Termination.Initiation of the chromosomal replication— 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 cell cycle— The prokaryotic versus eukaryotic cell cycleInitiation of the chromosomal replicationBacterial, Archaeal, Eukaryotic…— As always, …. bacteria … archaea … eukaryotes. — … similar complexity …at the forks… replication initiation … more complex in eukaryotes. — … simple in archaea, … … the multiple replication bubbles in eukaryotic chromosomes… (Kriegstein and Hogness, 1974)http://www.sciencedirect.com/science/article/pii/0022283673902234#http://nar.oxfordjournals.org/content/30/3/656/F3.expansionOrigin of replication, oriC-1— … Cairns: a single replication bubble in E. coli…... Prescott and Kuempel … both (sides) of the bubble… — … next … location of the replication origin… — … a synchronized culture … determine the timing of duplication of various points …. Replication forks!Origin of replication, oriC-2— Caro … a set of strains … single prophage Mu … around the chromosome. — … inserts randomly, … variously-positioned segment of homology, …— Another … probe … to a fixed position … prophage λ …— … dot-hybridization … the ratio of the hybridization signal at the variable position (Mu) to the signal at the constant position (λ), …. in the synchronized cultures, … λMu-1λMu-2λMu-3λMu-4λMu-5λMu-6Phage MuPhage λOrigin of replication, oriC-3— Caro thus determined the timing of the prophage Mu doubling… — … expectations …: 1) if initiation is random, then Mu:Lambda ratio will be stable, … — 2) if the initiation is at a specific point, … — … (collection of) strains … Mu …. different positions … gradient of times of Mu doubling relative to lambda… λMuλMuλMuMu/λ = 1ORMu/λ = 2 Mu/λ = 1/2Mu/λ ratioTime (generations)10Time (generations)10Mu/λ ratioOri-proximalOri-distalOrigin of replication, oriC-4— … synchronization of replication initiation. — …. two separate steps: 1) chromosome alignment, … 2) synchronous release … — … two distinct steps by changing a single variable… .— … (even though) antibiotics rifampicin or chloramphenicol … align the chromosomes, … the cell recovery takes hours... 12Origin of replication, oriC-5— … early way to synchronize…. block protein synthesis … required aminoacid …. —… finish ongoing replication rounds… — … all cells … single chromosome … (chromosomes become "aligned"). — When … allowed … missing aminoacid, … simultaneously initiated …1. Withdrawal of a required aminoacid2. Addition of the required aminoacidOrigin of replication, oriC-6— … synchronization is short-lived… in several hours … asynchronous …. — … the first couple of replication … synchronous. Time = 5' Time = 10' Time = 15' Time = 20'Origin of replication, oriC-7— … isolated chromosomal DNA at different time points… in a set of Lambda/Mu lysogens,… hybridization to determine kinetics of duplication …. — These are their much simplified results (“—” means the locus still single copy, “+” means the locus is duplicated):Mu Position DNA isolation time (min)Locus (Map minutes) 0 15 30 451 0 — + + +2 17 — — + +3 34 — — — +4 50 — — + +5 66 — + + +6 83 + + + +01734506683E. coli chromosomeOrigin of replication, oriC-8— … scenario #2: … signal doubling … location-specific.— … gradient of duplication … position 83 min… origin….. — … position 34 min … terminus.— … indeed bi-directional, … DNA isolation time (min)Locus Mu Position (Minutes) 0 15 30 451 0 — + + +2 17 — — + +3 34 — — — +4 50 — — + +5 66 — + + +6 83 + + + +017345066835'20'20'35'35'50'originterminusOrigin of replication, oriC-9— … culture synchronization was not necessary … dot hybridization… the maximal signal at the origin ….DNA isolation time (min)Locus Mu Position (Minutes) 0 15 30 451 0 — + + +2 17 — — + +3 34 — — — +4 50 — — + +5 66 — + + +6 83 + + + +origin terminusterminus12Origin / terminus = 2— … the typical ori/ter ratio = 2 is changed … oriterOrigin of replication, oriC-9—… oriC (for “origin of the chromosomal replication”). — … these days … genome arrays …. deep sequencing, reveal: 1) a wave of replication …;2) a perfect 2/1 ori/ter ratio … origin terminusterminus12origin terminusterminus12Origin / terminus = 25' 15' 25'Origin of replication, oriC-10— … archaeon Sulfolobus solfataricus has three replication origins … http://www.pnas.org/content/101/18/7046.longSulfolobus was isolated from Yellowstone hot springs.Origin of replication, oriC-11— Cloning of oriC … : 1) … short piece of DNA; 2) … well-tolerated by the cell. — … the chromosomal DNA … pieces with antibiotic-resistance genes … … one finds oriC. Chromosomal DNAAn antibiotic-resistance geneBreak randomlycombineoriCoriCoriCMini-chromosome— …. “minichromosomes”, even though … —… works reliably in lower eukaryotes, … autonomously-replicating sequences (ARSes) of a particular organism can be cloned by transformation … genomic library of this organism made in E. coli. … plasmids driven by ARSes of this organism.Eukaryotic replication origins… multiple, alternative…Do not worry, be happyDnaA and DnaC-1— … required aminoacid from … auxotrophic mutant and measures the rate … — …. ongoing rounds of DNA replication …— … (reminds)… tapering off … dnaA and dnaC. TCA-precipitable 3H-dT per 1'Time (minutes)0 10 20 30Removal of a required amino acidPrototrophic culture—… by their replication behavior, the delayed-stop mutants… initiation …— … biochemical properties…?DnaA and DnaC-2—DnaC protein …(binds)… DnaB. — … heterododecamer …: DnaB6DnaC6. — DnaC … dual role…? — …no
View Full Document
Unlocking...