Isolation of a dnaE mutation which enhances RecA‐independent homologous recombination in the Escherichia coli chromosome

Abstract

SummaryThe mechanism of recombination of tandem repeats in the chromosome of Escherichia coli was investigated by genetic means. Tandem repeats 624 bp long were introduced into the lacZ gene of E. coli and the efficiency of deletion of one repeat was compared in different recombination mutants. No effects of the recA, recBC, recF, ruvA or ruvA recG mutations were detected. Hence, tandem repeat deletion appears to not proceed via the RecBCD or RecF homologous recombination pathways. A new mutant in which RecA-independent recombination is increased 15-fold was isolated. The mutation lies in the dnaE gene coding for the alpha subunit of polymerase III: it is a Gly to Asp change at codon 133. Another dnaE mutation, dnaE486, was tested and also shown to stimulate RecA-independent recombination. It is proposed that tandem-repeat recombination occurs by a replication slippage mechanism. RecA-independent recombination is also enhanced in a rep mutant, in which chromosomal replication is slowed down by the absence of the Rep helicase, suggesting that replication pausing may facilitate slippage.

uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway

“…In a previous report, we described one such mutant, in which RecA-independent recombination was stimulated 15-fold. The mutation affects dnaE, the gene encoding the ␣-subunit of E. coli polymerase III (Bierne et al, 1997). Another dnaE mutation had a similar effect, as did a rep mutation, which inactivates a replicative helicase.…”

“…JJC520 was mutagenized with EMS at 30ЊC and plated on XPG plates at 37ЊC to isolate colonies with increased papillae. As described previously (Bierne et al, 1997), three thermosensitive (TS) and four UV S mutants showing increased deletion frequencies (hyperrecombination) were isolated. In a previous report, we described the characterization of one TS mutation, located in the dnaE gene encoding the ␣-subunit of DNA polymerase III.…”

“…Two resolution pathways have been described in E. coli depending on the ruvABC and recG loci respectively (Lloyd, 1991). ruvA, recG or a ruvA recG double mutation do not affect TRD in wild-type strains, in which TRD is RecA independent (Bierne et al, 1997 Bierne et al (1997).…”

“…The inactivation of either of the two main pathways for homologous recombination in E. coli, RecBCD and RecF, does not modify TRD frequency. Furthermore, these deletion events do not require a resolution step, as they do not cause dimerization of the chromosome and their frequency is unchanged in a ruvA recG double mutant, deficient for both E. coli resolution pathways (Bierne et al, 1997). Since, in wild-type strains, TRD does not seem to involve the formation or the resolution of a crossing over, it may occur by a new homologous recombination pathway.…”

“…Another dnaE mutation had a similar effect, as did a rep mutation, which inactivates a replicative helicase. These findings, suggestive of a replicative pathway of recombination, led us to propose that TRD occurs by replication slippage (Bierne et al, 1997). An independent study of TRD in various polymerase III mutants led to the same conclusion (Saveson and Lovett, 1997).…”