Abstract
Homologous recombination (HR) is crucial for the error-free repair of DNA double-strand breaks (DSBs) and the restart of stalled replication. However, imprecise HR can lead to genome instability, highlighting the importance of HR quality control. After DSB formation, HR proceeds via DNA end resection and recombinase loading, whereas helicase-catalyzed disruption of a subset of subsequently formed DNA invasions is thought to be essential for maintaining HR accuracy via inhibiting illegitimate (non-allelic) recombination. Here we show that in vitro characterized mechanistic aberrations of E. coli RecBCD (resection and recombinase loading) RecQ (multifunctional DNA-restructuring helicase) mutant enzyme variants, on one hand, cumulatively deteriorate cell survival under certain conditions of genomic stress. On the other hand, we find that RecBCD and RecQ defects functionally compensate each other in terms of HR accuracy. The abnormally long resection and unproductive recombinase loading activities of a mutant RecBCD complex (harboring the D1080A substitution in RecB) cause enhanced illegitimate recombination. However, this compromised HR-accuracy phenotype is suppressed in double mutant strains harboring mutant RecQ variants with abnormally enhanced helicase and inefficient invasion disruptase activities. These results frame an in vivo context for the interplay of biochemical activities leading to illegitimate recombination, and underscore its long-range genome instability effects manifest in higher eukaryotes.
Highlights
Homologous recombination (HR) is a biological process that occurs in all kingdoms of life, and provides the most efficient means for the error-free repair of DNA double-strand breaks (DSBs) and the restart of stalled DNA replication [1,2]
By using the RedET recombination system, we generated the following mutant E. coli strains expressing modified protein variants driven by their respective native promoters (S1 Table). recB1080 harbors a RecB protein with amino acid substitution D1080A, which has been shown to cause enhanced helicase activity of the RecB1080CD complex, combined with impaired nuclease and RecA-loading activities [5]
In the recQ-dH and recQ-dWH strains the protein is C-terminally truncated after aa 523 and 413, respectively, thereby lacking the HRDC and winged-helix (WHD) plus HRDC domains [15, 20].The RecQÃ and RecQ-dH proteins exhibit enhanced DNA unwinding activity, but lack the shuttling activity characteristic of RecQ-WT, and RecQ-dH is further severely impaired in its directed invasion disruption activity [15, 20]
Summary
Homologous recombination (HR) is a biological process that occurs in all kingdoms of life, and provides the most efficient means for the error-free repair of DNA double-strand breaks (DSBs) and the restart of stalled DNA replication [1,2]. HR is mediated by the resection of DSB ends, producing 3’ single-stranded (ss) DNA overhangs that are substrates for the subsequent formation of recombinase nucleoprotein (RNP) filaments. Program of the Hungarian Academy of Sciences (PPD-017/2017) to G.M.H
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