Abstract
It was recently reported that the recBC mutants of Escherichia coli, deficient for DNA double-strand break (DSB) repair, have a decreased copy number of their terminus region. We previously showed that this deficit resulted from DNA loss after post-replicative breakage of one of the two sister-chromosome termini at cell division. A viable cell and a dead cell devoid of terminus region were thus produced and, intriguingly, the reaction was transmitted to the following generations. Using genome marker frequency profiling and observation by microscopy of specific DNA loci within the terminus, we reveal here the origin of this phenomenon. We observed that terminus DNA loss was reduced in a recA mutant by the double-strand DNA degradation activity of RecBCD. The terminus-less cell produced at the first cell division was less prone to divide than the one produced at the next generation. DNA loss was not heritable if the chromosome was linearized in the terminus and occurred at chromosome termini that were unable to segregate after replication. We propose that in a recB mutant replication fork breakage results in the persistence of a linear DNA tail attached to a circular chromosome. Segregation of the linear and circular parts of this “σ-replicating chromosome” causes terminus DNA breakage during cell division. One daughter cell inherits a truncated linear chromosome and is not viable. The other inherits a circular chromosome attached to a linear tail ending in the chromosome terminus. Replication extends this tail, while degradation of its extremity results in terminus DNA loss. Repeated generation and segregation of new σ-replicating chromosomes explains the heritability of post-replicative breakage. Our results allow us to determine that in E. coli at each generation, 18% of cells are subject to replication fork breakage at dispersed, potentially random, chromosomal locations.
Highlights
The bidirectional replication of the Escherichia coli circular chromosome starts at the replication origin oriC and ends when forks meet in the opposite region, the chromosome terminus
We previously showed that this deficit results from heritable terminus DNA loss, owing to cell-division dependent double-strand break (DSB) in the chromosome terminus
It remains attached to the circular chromosome part, which triggers a DSB in the chromosome terminus during cell division in a heritable reaction
Summary
The bidirectional replication of the Escherichia coli circular chromosome starts at the replication origin oriC and ends when forks meet in the opposite region, the chromosome terminus. Replication forks are arrested in the terminus region by specific sites called ter where binding of the Tus protein blocks replication forks in an orientation-specific manner (reviewed in [1,2]). As chromosome segregation is concurrent with replication in bacteria, the origin and terminus regions are the first and the last DNA sequences to be segregated during chromosome partitioning [3,4,5]. The two origins first remain associated at mid-cell for about 20 min and move to the 1⁄4 and 3⁄4 positions of the cell. The terminus regions are replicated at mid-cell and only separate shortly before cell division [3,4,5]
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