The Extent of Migration of the Holliday Junction Is a Crucial Factor for Gene Conversion in Rhizobium etli

Abstract

Gene conversion, defined as the nonreciprocal transfer of DNA, is one result of homologous recombination. Three steps in recombination could give rise to gene conversion: (i) DNA synthesis for repair of the degraded segment, (ii) Holliday junction migration, leading to heteroduplex formation, and (iii) repair of mismatches in the heteroduplex. There are at least three proteins (RuvAB, RecG, and RadA) that participate in the second step. Their roles have been studied for homologous recombination, but evidence of their relative role in gene conversion is lacking. In this work, we showed the effect on gene conversion of mutations in ruvB, recG, and radA in Rhizobium etli, either alone or in combination, using a cointegration strategy previously developed in our laboratory. The results indicate that the RuvAB system is highly efficient for gene conversion, since its absence provokes smaller gene conversion segments than those in the wild type as well as a shift in the preferred position of conversion tracts. The RecG system possesses a dual role for gene conversion. Inactivation of recG leads to longer gene conversion tracts than those in the wild type, indicating that its activity may hinder heteroduplex extension. However, under circumstances where it is the only migration activity present (as in the ruvB radA double mutant), conversion segments can still be seen, indicating that RecG can also promote gene conversion. RadA is the least efficient system in R. etli but is still needed for the production of detectable gene conversion tracts.

Biased Gene Conversion in Rhizobium etli Is Caused by Preferential Double-Strand Breaks on One of the Recombining Homologs

“…This bias toward gain is particularly striking, taking into account that gene conversion events can encompass from 150 bp to 700 bp. All of these characteristics have been confirmed upon analyzing a larger sample, including cointegrates generated in mutants affected in recombination (4).…”

“…To prove this, we used a variation of a strategy described before (4,10). We have analyzed gene conversion in R. etli by using a genetic strategy (Fig.…”

“…3D]). Since this strategy does not select directly for gene conversion events, we consider it reliable enough for estimating frequencies and types of gene conversion (4,10). To deliver in vivo a double-strand cut on the incoming plasmid, the mobilizable plasmid with the RFLPs in nifH (pJGus28) was modified to include, in the middle of the gene, an 18-bp sector (labeled 1 in Fig.…”

“…Its genome, comprising one circular chromosome and six megaplasmids, has already been sequenced (2) and contains several repeated sequences, ranging from a few base pairs to several genes, that are at least 99% identical. These repeated sequences are substrates for recombination, which can lead to gene conversion (3)(4)(5)(6). Gene conversion is defined as the nonreciprocal transfer of information between two homologous sequences.…”

“…Nevertheless, given their well-detailed recombination genetics, availability of genome sequences, and the presence of many repeated sequences, bacterial and archaeal genomes are very interesting models for studying gene conversion (8,9). Previously, we have analyzed gene conversion in R. etli by using a genetic strategy in which the wild-type R. etli nifH gene was modified to include eight different restriction fragment length polymorphisms (RFLPs), spaced approximately every 100 bp along the gene (4,10). These RFLPs serve as markers for the extent and location of gene conversion events.…”