A new type of illegitimate recombination is dependent on restriction and homologous interaction

Abstract

Illegitimate (nonhomologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs. Under special conditions in Escherichia coli, we have found a new type of illegitimate recombination that requires an interaction between homologous DNA sequences. It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type II restriction in vitro and type I (EcoKI) restriction in vivo within a ⌬rac recBC recG ruvC strain. Removal of one of the repeats or its replacement with heterologous DNA resulted in a reduction in the level of recombination. The recombining sites themselves shared, at most, a few base pairs of homology. Many of the recombination events joined a site in one of the repeats with a site in another repeat. In two of the products, one of the recombining sites was at the end of one of the repeats. Removal of one of the EcoKI sites resulted in decreased recombination. We discuss the possibility that some structure made by homologous interaction between the long repeats is used by the EcoKI restriction enzyme to promote illegitimate recombination. The possible roles and consequences of this type of homologous interaction are discussed.Illegitimate recombination of DNA is defined as recombination between sequences containing little or no base pair sequence homology (34). The term is limited here to recombination processes taking place at low frequencies at numerous sites on the chromosome, as opposed to recombinational events at specific sites such as within immunoglobulin genes. The possible underlying mechanisms that have been proposed include the joining of double-stranded ends after degradation (47) and strand slippage during replication (1).Illegitimate recombination has been regarded as a process distinct from homologous recombination, which involves much longer regions of homology (typically in the kilobase range) (11) and depends on a different set of gene products (34). However, it has been suggested that some types of DNA lesions, such as double-strand DNA breaks, can trigger both illegitimate and legitimate (homologous) recombination (3).Analysis of aberrant homologous recombination products in mammalian cells has led to the proposal that interaction between two homologous DNA regions results in illegitimate recombination of one of the interacting sites with a third, uninvolved site (9,22,48). One of the possible molecular mechanisms that assumes the existence of a Holliday intermediate is illustrated in Fig. 1. According to this model, illegitimate recombination is a consequence of the destruction of an intermediate involved in homologous recombination, with some illegitimate recombination events possibly taking place at the ends of long regions of homology shared between the recombining DNAs. These illegitimate events have indeed been observed (9, 48).This model was able to explain several of the aberra...

DNA restriction is a barrier to natural transformation in Pseudomonas stutzeri JM300

“…Presently, only five RM systems have been detected in P. stutzeri (see http://rebase.neb.com), and it is not yet known how frequent and diverse RM systems are among the over 500 members of that species identified by molecular methods (Sikorski et al, 2002a, b). Several authors have pointed out that RM systems can contribute to an increase of genetic diversity in prokaryotes by allowing natural genetic engineering involving homologous and illegitimate recombination processes and by exerting certain selection pressure on the DNA sequence (Arber, 1991;Kusano et al, 1997;McKane & Milkman, 1995;Rocha et al, 2001). The data of this study add the notion that RM systems can contribute to speciation also by producing sexual isolation and thereby allowing the free divergence of the isolated lineage.…”

Homologous recombination at the border: Insertion-deletions and the trapping of foreign DNA in Streptococcus pneumoniae

“…NJs also were frequently found to be associated with microhomologies (29,31). Another example strikingly similar to the formation of InsDels in S. pneumoniae is that of the parahomologous targeting described in Drosophila cells (32).…”

“…Since our initial observation of homology-directed illegitimate recombination in S. pneumoniae (4), related phenomena have been reported, for example in E. coli (29) and yeast (30,31). NJs also were frequently found to be associated with microhomologies (29,31).…”

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

“…EcoKI, a well-studied type I enzyme, was shown to promote this homology-facilitated illegitimate recombination (127). In addition, there is evidence that R-M systems could bring in genome rearrangements (99,102,(128)(129)(130)(131)(132).…”

“…Restriction was also identified to play a role in nonhomologous recombination, in which a small stretch of homologous DNA sequences at one end is utilized to recombine and integrate a large foreign DNA (lacking homology) into recipient genomes (127). EcoKI, a well-studied type I enzyme, was shown to promote this homology-facilitated illegitimate recombination (127).…”