RecBCD, SbcCD and ExoI process a substrate created by convergent replisomes to complete DNA replication

Abstract

The accurate completion of DNA replication on the chromosome requires RecBCD and structure specific SbcCD and ExoI nucleases. However, the substrates and mechanism by which this reaction occurs remains unknown. Here we show that these completion enzymes operate on plasmid substrates containing two replisomes, but are not required for plasmids containing one replisome. Completion on the tworeplisome plasmids requires RecBCD, but does not require RecA and no broken intermediates accumulate in its absence, indicating that the completion reaction occurs normally in the absence of any double-strand breaks. Further, similar to the chromosome, we show that when the normal completion reaction is prevented, an aberrant RecA-mediated recombination process leads to amplifications that drive most of the instabilities associated with the two-replisome substrates. The observations imply that the substrate SbcCD, ExoI and RecBCD act upon in vivo is created specifically by two convergent replisomes, and demonstrate that the function of RecBCD in completing replication is independent of doublestrand break repair, and likely promotes joining of the strands of the convergent replication forks.

UV‐induced DNA damage disrupts the coordination between replication initiation, elongation and completion

“…Whereas RecF and nucleotide excision repair are required for replication to resume when it stops short, other gene products are required to complete or limit replication from going too far. The absence of RecBC, SbcCD, ExoI or RecG impairs the cell's ability to complete replication normally (Hamilton et al., 2019; Midgley‐Smith et al., 2018; Rudolph et al., 2013; Wendel et al., 2014, 2018). Both recBC and recG are hypersensitive to UV for reasons that remain unclear.…”

“…However, in the case of sbcCD xonA mutants, this phenotype is not associated with increased lethality, as cells recover and resume cell division (Figure 6c). In the absence of SbcCD and ExoI completion occurs through an aberrant pathway that is associated with amplifications and genomic instability, but retains viability (Hamilton et al., 2019; Wendel et al., 2018), perhaps accounting for the difference between this mutant and the UV‐hypersensitive recG and recBC . Consistent with this interpretation, whereas recBC and recG mutant cells remained filamentous for more than 4 hr after irradiation, sbcCD xonA mutants appeared filamentous at two hours after irradiation, but returned to normal by 4 hr after irradiation, suggesting that the chromosomal imbalances in this mutant can be resolved in a manner that allows survival (Figure S4).…”

The Roles of Bacterial DNA Double-Strand Break Repair Proteins in Chromosomal DNA Replication

“…Plasmid multimerization and instability in a recBC mutant have recently been re-examined and an attempt has been made to extrapolate from the behavior of plasmids to that of the chromosome (Wendel, Courcelle and Courcelle 2014 ; Hamilton et al . 2019 ). However, fork breakage, which triggers sigma replication of chromosomes in a recBC mutant, occurs in 18% of cells on a 4.6 Mb chromosome (discussed later in this review).…”

“…Clearly, such events occurring on a 5 kb plasmid present at 50–80 copies per cell would not be frequent enough to cause the observed growth impairment of a recBC mutant following transformation with a lambda-origin plasmid (Hamilton et al . 2019 ). Furthermore, the impact of nucleases on a 5 kb plasmid and on the chromosome are expected to be very different.…”

Under-Replicated DNA: The Byproduct of Large Genomes?

“…Terminating forks will certainly result in genomic instability if not resolved properly. In Escherichia coli , chromosome over-replication, deletions, and other DNA rearrangements are associated with defective replication termination [ 49 , 50 , 51 , 52 , 53 , 54 , 55 ]. Indeed, the termination region of the E. coli chromosome is considered a recombination hotspot [ 55 ].…”