Strand Specificity of Nicking of DNA at Chi Sites by RecBCD Enzyme

Abstract

RecBCD enzyme is essential for the major pathway of homologous recombination of linear DNA in Escherichia coli. It is a potent nuclease and helicase and, during its unwinding of double-stranded DNA, makes singlestrand scissions in the vicinity of Chi recombination hot spots. We report here that both the strand that is cut and the position of the cuts relative to Chi depended on the ATP to Mg 2؉ ratio. With ATP in excess, Chi-dependent nicks occurred, as we have previously reported, four to six nucleotides to the 3-side of the Chi octamer (5-GCTGGTGG-3) and were detected only on the strand bearing that sequence. Three differences were seen with Mg 2؉ in excess. 1) Chi-dependent 3-ends were produced on the GCTGGTGG-containing strand closer to and within the Chi octamer. 2) Chi-dependent cuts occurred on the complementary DNA strand. 3) RecBCD enzyme destroyed the 3-terminated strand of DNA from its entry point up to the vicinity of the Chi site, as others have previously reported. We show here that, with Mg 2؉ in excess, the enzyme continued to travel along DNA, after encountering a Chi site, releasing both strands of the DNA distal to Chi as single strands. We discuss potential biological consequences of these two modes of RecBCD enzyme-Chi interaction.RecBCD enzyme (EC 3.1.11.5), encoded by the recB, recC, and recD genes of Escherichia coli, is the best characterized enzyme specific to the major (RecBCD) pathway of homologous recombination in E. coli conjugation and transduction (reviewed in Ref. 1). Purified RecBCD has many activities. It has potent ATP-dependent ss 1 and ds DNA exonuclease activities and a weak ATP-stimulated endonuclease activity on ss, but not ds, circular DNA. Under conditions that reduce these nuclease activities (e.g. 5 mM ATP and 1 mM Mg 2ϩ ) the enzyme totally unwinds ds DNA, up to 40 kilobase pairs or longer in the presence of SSB (2-4). Electron microscopy revealed that the enzyme travels unidirectionally and highly processively from a ds DNA end through the DNA, with either transient or permanent unwinding of the DNA behind the enzyme (5, 6). Under such conditions the enzyme has a potent site-specific cleavage activity at Chi sequences (5Ј-GCTGGTGG-3Ј) (7), a recombinational hot spot specific to the RecBCD pathway (reviewed in Ref. 8). This cleavage occurs only as the enzyme is unwinding the DNA from right to left, as Chi is written here (9). The enzyme makes Chi-dependent ss nicks a few bases to the 3Ј-side of Chi on the GCTGGTGG-containing ("Upper") strand 2 but makes no detectable Chi-dependent nicks on the complementary strand (7, 9) (Fig. 1, left). The activities of the purified enzyme, under these conditions, correlate well with the properties of Chi deduced from genetic studies and support a previously proposed model of Chi-stimulated recombination by the RecBCD pathway (8, 10) (see "Discussion").RecBCD enzyme has been coupled with RecA and SSB proteins to produce Chi-dependent joint molecules from linear Chi-containing ds DNA and supercoiled Chi-free ds DNA (11)(12)(13). This...

Chi Hotspot Activity in Escherichia coli Without RecBCD Exonuclease Activity: Implications for the Mechanism of Recombination

“…Biochemical studies with purified proteins have demonstrated the activities of the individual enzymes noted above (reviewed by Smith 1988Smith , 2001Kowalczykowski et al 1994;Kowalczykowski 2000). Mutations that alter RecBCD or Chi coordinately alter Chi nicking Taylor et al 1985;Cheng and Smith 1987) or switching of DNA degradation from one strand to the other at Chi (Dixon and Kowalczykowski 1993;Taylor and Smith 1995b;Anderson and Kowalczykowski 1997a). These results are consistent with both models in Figure 1.…”

“…If the concentration of ATP is greater than that of Mg 21 ions (Figure 1, left), then RecBCD nicks the strand with Chi a few nucleotides to the 39 side of this sequence; continued unwinding produces a ssDNA fragment with Chi at its 39-end Taylor et al 1985;Taylor and Smith 1995b). If the concentration of Mg 21 is greater than that of ATP (Figure 1, right), RecBCD degrades the 39-ended strand up to the Chi sequence (Dixon and Kowalczykowski 1993), cuts the complementary strand (Taylor and Smith 1995b), and 1 continues to degrade that strand (in the 59 / 39 direction) (Anderson and Kowalczykowski 1997a); this reaction also produces ssDNA with Chi at or near its 39-end, called here the ''Chi tail.'' Thus, in the first reaction, Chi induces DNA cutting of the strand engaged by RecB to produce the Chi tail, whereas in the second reaction Chi blocks DNA cutting of that strand to produce the Chi tail.…”

Intersubunit signaling in RecBCD enzyme, a complex protein machine regulated by Chi hot spots

“…With excess ATP, this occurs by a simple nick (Taylor et al 1985), whereas with excess Mg 2+ , degradation of the 3Ј-ended strand ceases at or near Chi (Dixon and Kowalczykowski 1993;Taylor and Smith 1995b). The mutants studied here appear to generate 3Ј ends in a similar manner under these two reaction conditions ( Fig.…”

“…In reactions with excess Mg 2+ ions over ATP, RecBCD's exonuclease activity is high, and the enzyme degrades the 3Ј-ended strand up to Chi (Dixon and Kowalczykowski 1993), nicks the complementary strand (Taylor and Smith 1995b), and then degrades the 5Ј-ended strand during continued unwinding (Anderson and Kowalczykowski 1997a). At least under this condition, the enzyme begins to load RecA protein onto the 3Ј-ended strand to the left ("downstream") of Chi (Anderson and Kowalczykowski 1997b), and the single-stranded DNA (ssDNA)-RecA fila- Figure 1.…”

An Overview on Molecular Basis of Genetic Recombination

“…Second, all recombination stimulated by this site requires the activity of the RecBCD enzyme, whose nuclease activity would seemingly destroy its own substrate. Numerous genetic and biochemical studies have shown that the increase in recombination is due to a direct interaction between the x sequence and the RecBCD enzyme, and that this stimulation only occurs if the enzyme approaches from the 3" side (Figure 4) (Taylor and Smith, 1995). The interaction with x elicits several changes in enzyme function that are manifest in an overall decrease in nuclease activity, which accounts for the protection of DNA observed in vivo.…”