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Role for DNA double strand end-resection activity of RecBCD in control of aberrant chromosomal replication initiation in Escherichia coli

Sayantani Goswami

Published 2022 in bioRxiv

ABSTRACT

Replication of the circular bacterial chromosome is initiated from a locus oriC with the aid of an essential protein DnaA. One approach to identify factors acting to prevent aberrant oriC-independent replication initiation has been that to obtain mutants which survive loss of DnaA. Here we show that a ΔrecD mutation, associated with loss of RecBCD’s double strand-DNA exonuclease V activity, provokes abnormal replication and rescues ΔdnaA lethality in two situations: (i) in absence of 5’-3’ single-strand DNA exonuclease RecJ, or (ii) when multiple two-ended DNA double strand breaks (DSBs) are generated either by I-SceI endonucleolytic cleavages or by radiomimetic agents phleomycin or bleomycin. With two-ended DSBs in a ΔrecD strain, ΔdnaA viability was retained even after linearization of the chromosome. Data from genome-wide DNA copy number determinations in ΔdnaA-rescued cells lead us to propose a model that exonuclease V-mediated DNA resection activity is critical for prevention of a σ-mode of rolling-circle over-replication when convergent replication forks merge and fuse; such mergers occur during normal replication at the chromosomal terminus region, as well as during repair of two-ended DSBs following “ends-in” replication. Resection mechanisms may also be important in eukaryotes to prevent DNA over-replication at sites of fork merger.

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