RecA-independent recombination is efficient but limited by exonucleases

Abstract

Genetic recombination in bacteria is facilitated by the RecA strand transfer protein and strongly depends on the homology between interacting sequences. RecA-independent recombination is detectable but occurs at extremely low frequencies and is less responsive to the extent of homology. In this article, we show that RecAindependent recombination in Escherichia coli is depressed by the redundant action of single-strand exonucleases. In the absence of multiple single-strand exonucleases, the efficiency of RecA-independent recombination events, involving either gene conversion or crossing-over, is markedly increased to levels rivaling RecAdependent events. This finding suggests that RecA-independent recombination is not intrinsically inefficient but is limited by single-strand DNA substrate availability. Crossing-over is inhibited by exonucleases ExoI, ExoVII, ExoX, and RecJ, whereas only ExoI and RecJ abort gene-conversion events. In ExoI ؊ RecJ ؊ strains, gene conversion can be accomplished by transformation of short single-strand DNA oligonucleotides and is more efficient when the oligonucleotide is complementary to the lagging-strand replication template. We propose that E. coli encodes an unknown mechanism for RecA-independent recombination (independent of prophage recombination systems) that is targeted to replication forks. The potential of RecA-independent recombination to mediate exchange at short homologies suggests that it may contribute significantly to genomic change in bacteria, especially in species with reduced cellular exonuclease activity or those that encode DNA protection factors.DNA rearrangements ͉ DNA repair ͉ genetic recombination ͉ genetic exchange ͉ replication fork repair T he recA gene was the first gene discovered to mediate homologous recombination (1) and is highly conserved in bacteria (2). Archaea and eukaryotes also encode structurally and functionally related proteins, RadA and Rad51, respectively, that are similarly required for genetic recombination and DNA repair (3-6). RecA protein forms a helical filament on single-strand DNA (ssDNA) and catalyzes strand pairing and transfer between homologous DNA molecules in vitro, processes that initiate homologous recombination (reviewed in ref. 7). In Escherichia coli, where its function has been best studied, mutants in recA show severe reductions in recombination measured by different types of genetic crosses (reviewed in ref. 8).ssDNA initiates recombination, and its coating by RecA also likely protects it from nucleolytic attack. E. coli possesses four potent exonucleases (ExoI, ExoVII, ExoX, and RecJ) that digest ssDNA. Three of these exonucleases (RecJ, ExoI, and ExoVII) are processive enzymes, and all four exonucleases can degrade DNA at the approximate rate of thousands of bases per min (9-15). ExoI and ExoX degrade DNA strictly in the 3Ј to 5Ј direction, RecJ digests 5Ј to 3Ј, and ExoVII can degrade a strand of either polarity.Our previous work suggested that the ssDNA exonucleases (ssExos) of E. coli redundantly abort a number ...

Bacterial natural transformation by highly fragmented and damaged DNA

“…S1 for characterization of these A. baylyi mutants) revealed that singlestrand DNA exonucleases are the main factors degrading short cytoplasmic DNA (Fig. 1C and SI Text), consistent with reports on limiting factors for artificial transformation by electroporation (18,19). Experiments with different DNA concentrations showed that transformation by 60-bp substrates resulted in one-hit kinetics, as is the case with large molecules (SI Text and Fig.…”

“…1C) with DNA fragments occurred by single-strand annealing of the incoming DNA strand with the discontinuously synthesized strand at replication forks (Fig. 3A), as previously described after artificial DNA exposure in Escherichia coli and Legionella pneumophila (18,19). We confirmed this experimentally by using short mismatch-containing heteroduplex donor DNA and sequencing of transformants (Fig.…”

“…Fragmented and chemically damaged DNA is recognized as an important microbial nutrient source but has so far not been considered to contribute to bacterial genome evolution (1,(18)(19)(20). We decided to assess whether bacterial cells can acquire such degraded DNA, including genetic signatures of the deep past, by horizontal gene transfer-a driving force in prokaryotic evolution (21)(22)(23)(24).…”

Oligonucleotide recombination: A hidden treasure

“…These suppositions are supported by studies of mutant strains of Escherichia coli, lacking combinations of nucleases, that are capable of elevated levels of recombination with linear DNA. 7,8 These studies are informative and useful in some cases, but not universally applicable because of the requirement that experiments be performed in mutant strains.…”

Oligonucleotide transformation of yeast reveals mismatch repair complexes to be differentially active on DNA replication strands

“…This process is RecA-independent and reddependent, requiring only the ␤-protein (5). Recent work suggests that the ␤-protein is not required in the absence of ExoI and RecJ (6). A key discovery was the finding that MMR decreased transformation efficiency by as much as 100-fold, depending on the ssOligo sequence.…”