Academic journal article Genetics

RecBCD Enzyme "Chi Recognition" Mutants Recognize Chi Recombination Hotspots in the Right DNA Context

Academic journal article Genetics

RecBCD Enzyme "Chi Recognition" Mutants Recognize Chi Recombination Hotspots in the Right DNA Context

Article excerpt

HOMOLOGOUS recombination, like other aspects of chromosome metabolism, is controlled by special DNA sites. The sites controlling homologous recombination that are best understood at the molecular level are the Chi hotspots of Escherichia coli (Smith 2012). Chi sites control RecBCD enzyme, a complex enzyme with both nuclease and helicase activities essential for the major pathway of DNA double-strand break repair and genetic recombination. These sites, called Chi for crossover hotspot instigator (Lam et al. 1974), locally stimulate recombination by altering the multiple activities of RecBCD. Here, we address the question of how RecBCD recognizes Chi, the first step in its stimulation of recombination. Our results force a reanalysis of both the Chi nucleotide sequence and how RecBCD recognizes Chi.

Chi was discovered as a set of mutations that increase the plaque size of phage l red gam mutants, which rely on the E. coli RecBCD pathway for growth (Lam et al. 1974; McMilin et al. 1974; Henderson and Weil 1975). In the absence of its own recombination functions (Red) and of the RecBCD inhibitor (Gam), l replication forms only monomeric circles. Phage DNA packaging, however, requires dimeric or higher order concatemers, which can be formed by RecBCDpromoted recombination between monomers. Wild-type l lacks Chi sites; thus, the RecBCD pathway operates on l red gam mutants only at low level, only a few packaged phage are produced, and burst and plaque sizes are small. Mutations creating a Chi hotspot arise at multiple, widely scattered locations in the genome (Henderson and Weil 1975; Stahl et al. 1975); increase the frequency of RecBCD-promoted concatemer formation; and result in larger burst and plaque sizes. Analysis of six Chi sites in l and one in the E. coli lacZ gene revealed a sequence, 59 GCTGGTGG 39,commontoall (Smith et al. 1984). Mutations creating or inactivating Chi are all in this octamer. The flanking sequences have no discernible similarity, and it was concluded that Chi is 59 GCTGGTGG 39, its complement, or the duplex (Smith et al. 1981a). Indeed, insertion of synthetic DNA with 59 GCTGGTGG 39 results in Chi activity both with purified enzyme and in cells (Dixon and Kowalczykowski 1991; Dabert et al. 1992; Dabert and Smith 1997). Analysis of heteroduplex DNA showed that only the strand with 59 GCTGGTGG 39 is needed to activate purified RecBCD (Bianco and Kowalczykowski 1997).

Since Chi acts on the host RecBCD pathway but not on the l Red pathway or on two host pathways activated by suppressors of recBC null mutations (Gillen and Clark 1974; Stahl and Stahl 1977), it seemed likely that Chi interacts with RecBCD enzyme, the component unique to the RecBCD pathway. This hypothesis was bolstered by the isolation of intragenic pseudorevertants of a recC null mutant that regained at least partial recombination proficiency but lacked detectable Chi activity (Schultz et al. 1983).

Direct evidence that RecBCD recognizes Chi came from the demonstration that wild-type RecBCD enzyme, but not that from the pseudorevertants, nicks DNA at Chi during DNA unwinding (Ponticelli et al. 1985; Taylor et al. 1985). Collectively, these observations supported a model of recombination (Figure 1A) (Smith et al. 1981b) in which RecBCD initiates DNA unwinding at a free double-stranded (ds) DNA end, rapidly unwinds the DNA with the production of single-stranded (ss) DNA loops, nicks one strand at a properly oriented Chi site, and continues unwinding. The newly generated 39 ss end, with Chi near its end, is coated by RecA strand-exchange protein and invades an intact homologous duplex to generate a D-loop. The D-loop is converted into a Holliday junction, which may be resolved into reciprocal recombinants; alternatively, the D-loop may prime DNA replication and generate nonreciprocal recombinants (Smith 1991, 2012). Later reports showed that RecBCD actively loads RecA onto the newly generated 39 end in a Chi-dependent manner (Anderson and Kowalczykowski 1997). …

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