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Sunday, May 11, 2008

DNA Replication in E. coli: The Solution

In an earlier posting I described a problem that we often use to encourage critical thinking in our undergraduates. The problem is how can E. coli divide faster that the time it takes to replicate it's chromosome? [DNA Replication in E. coli: The Problem]

Recall that DNA replication always begins at an origin of replication. In bacteria there is usually one origin per chromosome or plasmid. (Eukaryotic chromsomes have multiple origins.)

The replisomes assemble at the origin and then move in opposite directions around the chromomome until the meet at the termination region. Each replisome moves at a rate of 1000 nucleotides per second and it takes about 38 minutes to complete one round of replication. But E. coli can divide every 20 minutes. That's the problem.

The firing of an origin is controlled by regulatory proteins. These proteins trigger the assembly of replisomes at the origin sequences. When most of us are first presented with this problem we think in terms of the events occurring sequentially. Thus, a chromosome is copied, the daughter chromosomes segregate, and a new round of replication begins.

This isn't what happens when the cells are dividing rapidly. Instead, a new round of replication begins at the "future origin" before the current round of replication is completed. At any given instant, there can be six or eight replication forks synthesizing DNA simultaneously inside the cell.

In order for the cell to divide every 20 minutes, all that is required is that a round of replication terminate every 20 minutes. This means that origins fire every 20 minutes. When the daughter chromosome segregate into daughter cells, they are already partially replicated in preparation for the next cell division.

Here's how Fossum et al. (2007) describe the solution in a recent paper in EMBO Journal.
The bacterium Escherichia coli has a single chromosome that is replicated from a single origin (oriC), bidirectionally to the terminus, once per division cycle (Kornberg and Baker, 1992). The cell cycle of slowly growing bacteria is quite similar to that of eukaryotic cells (Boye et al, 1996), with the G1, S and G2/M phases of bacteria termed B, C and D, respectively. E. coli (and certain other bacteria) is capable of very rapid growth in rich medium, with doubling times as short as 20 min. The replication time, however, remains long, with approximately 60–90 min required to replicate and segregate the chromosome. Therefore, the cell cycle is more complicated during rapid growth (Figure 1). If the time it takes to synthesize and segregate the daughter chromosomes (C+D) exceeds one generation time, a new round of replication must be initiated before the previous round is completed (Cooper and Helmstetter, 1968). Thus, initiation occurs at two origins in the 'mother' cell. It can even occur in the 'grandmother' cell at four origins if the time it takes to replicate and segregate the chromosome exceeds two generations. These initiations at two or four origins occur simultaneously, as one event per division cycle (Skarstad et al, 1986). While E. coli and Bacillus subtilis are two examples of bacteria capable of performing multifork replication, other bacteria, such as Caulobacter crescentus, are not. Eukaryotic cells do not replicate with overlapping cycles, but do initiate DNA replication at multiple replication origins, and thus perform a different kind of multifork replication, with the multiple forks on the same copy of the genome (Diffley, 2004).

Figure 1:Replication pattern of rapidly growing E. coli wild-type cells. Cells (yellow) with chromosomes (blue lines) and origins (black squares) are drawn schematically to show the number of replication forks and origins at different stages of the cell cycle. In this example, initiation of replication occurs at four origins at the same time as cell division (bottom). A young cell therefore contains four origins and six replication forks (upper left). As replication proceeds, the oldest pair of forks reach the terminus and the two sister chromosomes segregate. The cell then contains four origins and four replication forks (upper right). Initiation then occurs again at 4 origins and generates 8 new forks giving a total of 12 forks, as cell division approaches (bottom). Because there will be cell-to-cell variability, some cells will contain eight origins before they divide, whereas cells that divide before initiation of replication will contain only two origins (not shown). However, the majority of the cells in the culture will contain four origins.

Fossum, S., Crooke, E. and Skarstad, K. (2007) Organization of sister origins and replisomes during multifork DNA replication in Escherichia coli. EMBO J 26:4514–4522 [doi:10.1038/sj.emboj.7601871]


Anonymous said...

Ah, I guessed right. But doesn't this mean that the DNA is also doing its 'regular' job of being a template for RNA at the same time as it is replicating?
Why do none of the micrographs showing replication also show this?

Anonymous said...

Well, you had me beat there Larry. Thanks for the elegant explanation.
The consequential question, is of course; If you have several replications of DNA going on simultaneously, do you therefore have groups of cells (in twos, fours or eights) that have not completely budded off the 'mother' cell? Is this kind of replication common?

Torbjörn Larsson said...

Duh, that Sunday. Forgot to check the date on the previous post.

Now I won't know if I guessed right from scratch or happened to view the solution figures when scrolling down to the first unread post... [Checking: Yes, they are visible from the main page. Bummer!]

But at least I got the variation problem, if not the regulatory solution, correct. Yay!

TheChemistryOfBeer said...

Larry, this reminds me of the Star Trek Trouble with Tribbles episode .

Anonymous said...

Next step...
Consider which genes tend to be located nearer the replication origins and why this might be.

Torbjörn Larsson said...
This comment has been removed by the author.
Torbjörn Larsson said...

That seems to be what they call a good question! (As the answer, trivial as it may be, is rewarding.)