Crossing Over Between Regions of Limited Homology in Escherichia coli: RecA-Dependent and RecA-Independent Pathways

Abstract

We have developed an assay for intermolecular crossing over between circular plasmids carrying variable amounts of homology. Screens of Escherichia coli mutants demonstrated that known recombination functions can only partially account for the observed recombination. Recombination rates increased three to four orders of magnitude as homology rose from 25 to 411 bp. Loss of recA blocked most recombination; however, RecA-independent crossing over predominated at 25 bp and could be detected at all homology lengths. Products of recA-independent recombination were reciprocal in nature. This suggests that RecA-independent recombination may involve a true break-and-join mechanism, but the genetic basis for this mechanism remains unknown. RecA-dependent crossing over occurred primarily by the RecF pathway but considerable recombination occurred independent of both RecF and RecBCD. In many respects, the genetic dependence of RecA-dependent crossing over resembled that reported for single-strand gap repair. Surprisingly, ruvC mutants, in both recA+ and recA mutant backgrounds, scored as hyperrecombinational. This may occur because RuvC preferentially resolves Holliday junction intermediates, critical to both RecA-dependent and RecA-independent mechanisms, to the noncrossover configuration. Levels of crossing over were increased by defects in DnaB helicase and by oxidative damage, showing that damaged DNA or stalled replication can initiate genetic recombination.

The rarA gene as part of an expanded RecFOR recombination pathway: Negative epistasis and synthetic lethality with ruvB, recG, and recQ

“…However, it is not known how often postreplication gaps are formed, how they are formed, how large they are, or what types of lesions are most proficient in triggering their formation. There appear to be three major paths for filling post-replication gaps in bacteria: (a) RecA-mediated homologous recombination [16][17][18][19], (b) translesion DNA synthesis [9,17,20], and (c) a RecA-independent template switching process [21][22][23][24]. Based on the effects of gene inactivation, RecA-mediated homologous recombination is probably the most important of these processes.…”

“…The third pathway for post-replication gap filling is RecA-independent template switching. This process has been documented by examination of recombination events between relatively short, repeated DNA sequences [ 21 , 22 , 90 , 91 ]. Although most of the homologous recombination in bacteria is carried out by RecA recombinase, a measurable level of recombination events has been documented in ΔrecA cells [ 21 , 22 , 90 , 91 ].…”

“…This process has been documented by examination of recombination events between relatively short, repeated DNA sequences [ 21 , 22 , 90 , 91 ]. Although most of the homologous recombination in bacteria is carried out by RecA recombinase, a measurable level of recombination events has been documented in ΔrecA cells [ 21 , 22 , 90 , 91 ]. This homology-dependent but RecA-independent recombination is increased in cells with defective DNA replication and restart pathways [ 24 , 92 ].…”

Escherichia coli BW25113 Competent Cells Prepared Using a Simple Chemical Method Have Unmatched Transformation and Cloning Efficiencies

“…Further, recA + significantly increased the cloning efficiency ( Figures 5 , 8 ) but did not affect the stability of inserts with tandem repeats of various size and a 3-kb host DNA ( Figures 5 , 7 , 8 ). Considering the rate of RecA-dependent homologous recombination in E. coli is very low, occurring at approximately 10 −5 per cell generation ( Lovett et al, 2002 ), the effect of RecA on cloned inserts may be tolerated. Indeed, the recA + E. coli BL21(DE3) is routinely used in protein overexpression ( Chan et al, 2013 ), and recA + K-12 derivatives are widely used in metabolic engineering ( Cui et al, 2018 ; Badri et al, 2021 ).…”

“…Indeed, the recA + E. coli BL21(DE3) is routinely used in protein overexpression ( Chan et al, 2013 ), and recA + K-12 derivatives are widely used in metabolic engineering ( Cui et al, 2018 ; Badri et al, 2021 ). Hence, the effect of RecA on insert stability will not affect the cloned inserts in most cases, especially when homologous regions are small or not present ( Lovett et al, 2002 ).…”