1 MCB 502A-2015. Lecture #10. Initiation and Termination. Initiation of the chromosomal replication Bacterial origin of replication, oriC As always, we will start with bacteria and then will see how much more complex replication initiation is in archaea and eukaryotes. In fact, in contrast to the similar complexity of replication fork structure/function, replication initiation is significantly more complex in eukaryotes. Since it is quite simple in archaea, even though it is mechanistically related to the eukaryotic one, the complexity of eukaryotes is likely due to the multiple replication bubbles in their chromosomes, compared to the single bubble of prokaryotes. Recall the seminal demonstration of Cairns of a single replication bubble in E. coli, corroborated by the later demonstration by Prescott and Kuempel that both replication forks of the bubble are active. The next question in that line of research was about the location of the replication origin: is it fixed or is it random? This could have been done in a synchronized culture (where all the cells begin chromosomal replication at the same time) if one can determine the timing of duplication of various points along the chromosome. Caro decided to address this question with a set of strains each harboring a single prophage Mu inserted at various (mapped) locations around the chromosome. The Mu prophage, that inserts randomly, served as a variously-positioned segment of homology, to which a radioactive probe could be hybridized. Another differently-radioactive probe was hybridized to a fixed position on the chromosome, represented by prophage lambda that inserts at its fixed att site. By using dot-hybridization to determine the ratio of the hybridization signal at the variable position (Mu) to the signal at the constant position (lambda), it was possible to tell, in the synchronized cultures, when the duplication of a particular chromosomal point has occurred. Caro thus determined the timing of the prophage Mu doubling in all these strains after the initiation of replication. The two expectations were: 1) if initiation is random, then doubling of the Mu:Lambda ratio will never be observed, because the signal from cells in which Mu replicated first will be compensated by the signal from cells in which Lambda replicated first; 2) if the initiation is at a specific point, in every strain the doubling of the probe will be rapid but will happen only around a specific time after initiation. Comparison of all these strains in which Mu resides in different positions around the chromosome should produce a gradient of times of Mu doubling relative to lambda, increasing away from the origin. Making all cells in the culture initiate replication at the same time is called synchronization of replication initiation. Replication synchronization has two separate steps: 1) chromosome alignment, when the ongoing replication rounds are allowed to finish, but new initiations are blocked; 2) synchronous release of the initiation block. The most useful techniques achieve these two distinct steps by changing a single variable in the least invasive way. For example, antibiotics rifampicin or chloramphenicol completely block new initiations and aligns the chromosomes, but upon antibiotic removal, the cell recovery takes hours, so no replication synchrony can be achieved this way. A popular early way to synchronize chromosomal replication in E. coli was to block protein synthesis by withdrawing a required aminoacid from an auxotrophic mutant. Cells in which protein synthesis is blocked, finish ongoing replication rounds but do not initiate new rounds. Eventually, all cells in the amino acid-starved culture contain a single chromosome without replication bubbles (chromosomes become "aligned"). When protein synthesis is again2 allowed by addition of the missing aminoacid, the chromosomal replication is more-or-less simultaneously initiated in all cells of the culture. Such a replication synchronization is short-lived, of course, and in several hours the culture will be perfectly asynchronous again. However, during the first couple of replication cycle we may consider replication as being synchronous. Caro and colleagues isolated chromosomal DNA at different time points after the synchronous initiation of chromosomal replication in a set of Lambda lysogens, each of them also carrying a Mu insert at a distinct (known) position around the chromosome, and used hybridization to determine kinetics of duplication of the Mu prophage locus (relative to Lambda) in all these strains. These are their much simplified results (“—” means the locus still single copy, “+” means the locus is duplicated): What they found was scenario #2: the timing of signal doubling at various locations was location-specific, with a gradient of duplication timing pointing towards one particular location, position 83 min, as the replication origin (the first to replicate chromosome location). In the same study, they also found that culture synchronization was not necessary to locate the origin, as dot hybridization was linear in determining the copy number of loci around the chromosome in freely-replicating cultures and had the maximal signal at the origin and minimal at the opposite side of the chromosome. This origin of chromosomal replication is called oriC (for “origin of the chromosomal replication”). The same experiments, performed these days by hybridization to genome arrays or, more recently, deep sequencing, reveal: 1) a wave of replication spreading around the chromosome from the origin in synchronized cultures; 2) a perfect 2/1 ori/ter ratio in asynchronous cultures (replication time = division time), with the apex at the replication origin. As an exception among prokaryotes, archaeon Sulfolobus solfataricus has three replication origins in its circular chromosome, and so the gene array profiles of asynchronous chromosomal replication feature three peaks and three troughs (positions where replication forks meet). Cloning of oriC was facilitated by its two characteristics: 1) oriC is a relatively short piece of DNA; 2) several copies of the oriC sequence are well-tolerated by the cell. Indeed, if one cuts the chromosomal DNA with restriction enzymes, combines the resulting pieces with antibiotic-resistance genes and selects for autonomously-replicating antibiotic-resistance plasmids, one finds oriC. Such oriC-driven small plasmids are
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