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.