Replication Fork Breakage and Restart in Escherichia coli

Abstract

n all organisms, replication impairments are an important source of ge-nome rearrangements, mainly because of the formation of double-stranded DNA(dsDNA) ends at inactivated replication forks. Three reactions for the formation ofdsDNA ends at replication forks were originally described forEscherichia coliand be-came seminal models for all organisms: the encounter of replication forks with pre-existing single-stranded DNA (ssDNA) interruptions, replication fork reversal, andhead-to-tail collisions of successive replication rounds. Here, we first review the ex-perimental evidence that now allows us to know when, where, and how these threedifferent reactions occur inE. coli. Next, we recall our recent studies showing that inwild-typeE. coli, spontaneous replication fork breakage occurs in 18% of cells ateach generation. We propose that it results from the replication of preexisting nicksor gaps, since it does not involve replication fork reversal or head-to-tail fork colli-sions. In therecBmutant, deficient for double-strand break (DSB) repair, fork break-age triggers DSBs in the chromosome terminus during cell division, a reaction that isheritable for several generations. Finally, we recapitulate several observations sug-gesting that restart from intact inactivated replication forks and restart from recom-bination intermediates require different sets of enzymatic activities. The finding that18% of cells suffer replication fork breakage suggests that DNA remains intact at most inactivated forks. Similarly, only 18% of cells need the helicase loader for repli-cation restart, which leads us to speculate that the replicative helicase remains onDNA at intact inactivated replication forks and is reactivated by the replication re-start proteins.