Reversible inactivation of the Escherichia coli RecBCD enzyme by the recombination hotspot chi in vitro: evidence for functional inactivation or loss of the RecD subunit.

Abstract

Genetic recombination in Escherichia coil is stimulated by a RecBCD enzyme-mediated event at DNA sequences known as Chi (X) sites (5'-GCTGGTGG-3'). Previously, it was shown that X acts to regulate the nuclease activity of RecBCD; here, we demonstrate that, under appropriate conditions, interaction with X sites can also result in an inactivation of helicase activity of RecBCD (ssDNA) and double-stranded DNA (dsDNA) by means of its ATP-dependent exonuclease activities and its ATP-stimulated ssDNA endonuclease activity. In addition to these degradative activities, the enzyme is a DNA helicase that can processively unwind large tracts of dsDNA (>30 kb) at rates up to 1000 bp/sec (2-4). During dsDNA unwinding, the ATP-dependent degradation ofDNA is asymmetric, with the 3'-terminal DNA strand at the entry site for RecBCD being degraded more vigorously than the 5'-terminal strand (5-8).The X recombination hotspots are composed of the DNA sequence 5'-GCTGGTGG-3' (9, 10); recombination events are 5-10 times more frequent near X than far from it and these exchanges are recBC-dependent (11-13). Stimulation of recombination by X occurs primarily downstream (5' side) of the X site and extends, with decreasing magnitude, for >10 kb from X (12, 14). In vitro, recognition of X by RecBCD results in a single-strand break in the DNA strand containing the X sequence, 4-6 nucleotides to the 3' side of the X sequence; this occurs during the unwinding ofdsDNA and is orientation dependent (15, 16). These properties led to models which proposed the interaction between RecBCD and X served to initiate the RecA-dependent DNA strand invasion (17, 18).Recently, the formation of kspecific homologously paired joint molecules was demonstrated in vitro using a reconstituted recombination reaction consisting of purified RecA, RecBCD, and ssDNA-binding protein (SSB) (7). These results supported most predictions of the nick-initiation model (18), but they also uncovered an added feature of the RecBCD-X interaction (7,8). X recognition resulted in attenuation of the nuclease, but not the helicase, activity of RecBCD (7, 8 The biochemical basis for this x-dependent attenuation of nuclease activity remained unknown; however, the interpretations of genetic experiments propose a testable hypothesis (19,20). To explain the hyper-recombination phenotype of recD null mutations, it was proposed that interaction with a X site activates the nonrecombinogenic RecBCD by the removal of the RecD subunit, producing a RecBC(D-) protein that is changed to become a recombinogenic resolver of Holliday junctions (19,20). In this report, we describe experiments which test the RecD subunit-removal concept. We demonstrate that during the process of DNA unwinding at conditions of low nuclease activity (i.e., low Mg2+ concentration), the specific protein-DNA interaction between RecBCD and X causes a reversible inactivation of RecBCD's helicase activity and, we presume, its nuclease activity as well. This x-specific inhibition is fully reversible, and all RecBCD ac...

The RecD subunit of the Escherichia coli RecBCD enzyme inhibits RecA loading, homologous recombination, and DNA repair

“…activity. RecBCD enzyme recognizes Chi (4) and produces a 3Ј end for RecA loading by nicking the DNA (4, 5) or reducing its degradative activity (10 (8,9,28,46). This result indicates that RecB D1080A CD enzyme recognizes Chi; lacking nuclease activity, however, the enzyme fails to cleave the DNA (27) or load RecA (28).…”

A Single Mutation, RecBD1080A, Eliminates RecA Protein Loading but Not Chi Recognition by RecBCD Enzyme

“…Inactivation of RecBCD enzyme in response to at low concentrations of Mg 2ϩ does not cause the enzyme to dissociate from the DNA at (32). Rather, RecBCD enzyme continues to process the DNA until it reaches the end of the DNA molecule; however, this -modified enzyme is unable to re-initiate unwinding on another DNA molecule.…”

“…Nevertheless, in the absence of -specific fragment formation, it is still possible to detect recognition using the phenomenon of -dependent inactivation. Under conditions of low free Mg 2ϩ , wild-type RecBCD enzyme is inactivated after encountering a site while translocating through dsDNA (32). Therefore, the rate and extent for unwinding of -containing DNA by the RecB D1080A CD enzyme was examined at low free Mg 2ϩ concentrations.…”

Chi‐activated RecBCD enzyme possesses 5′→3′ nucleolytic activity, but RecBC enzyme does not: evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit

“…Rather, we propose that x-recognition results in an alteration of the RecD subunit function, which is reflected in both a x-dependent inactivation of initiation of helicase activity (i.e. reversible inactivation, Dixon et al 1994) and a x-dependent activation of nuclease activity (5 0 →3 0 , Anderson & Kowalczykowski 1997). Intriguingly, the RecD protein contains a sequence that has homology to several 5 0 →3 0 specific DNA exonucleases (Sue Lovett, Braudeis University, MA, personal communication).…”