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Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA
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Abstract |
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[Keywords: Homologous recombination; E. coli; RecBCD enzyme; Chi sites; complex protein machines]]
Received August 17, 2007; revised version accepted October 16, 2007.
). The multiple activities of each machine must be properly regulated for the process to be successful, but the basis of the regulation is in many cases unclear. We present here experimental results leading to a new hypothesis for how one such machine, the RecBCD enzyme of Escherichia coli, is regulated by Chi, a special nucleotide sequence in the DNA on which RecBCD acts during genetic recombination and the repair of DNA double-strand breaks (DSBs). The faithful repair of DSBs is crucial for living cells. Failure to repair such breaks can result in the loss of genetic information, and incorrect repair can yield deleterious genome rearrangements. Repair using homologous but genetically different DNA as a template can produce genetic recombinants, thereby increasing genetic diversity and aiding evolution. Recombination thus provides both short-term and long-term benefits to living organisms.
Recombination is a complex process that requires multiple proteins and enzymatic activities. At the molecular level, one of the best understood paradigms is the major (RecBCD) pathway of DSB repair and recombination in E. coli. Essential for this pathway is the RecBCD enzyme, a protein machine with multiple activities on DNA that promote the initial stages of recombination (Smith 2001). These multiple activities are regulated by Chi sites, 5'-GCTGGTGG-3', which are hot spots of recombination by the RecBCD pathway (Stahl and Stahl 1977). The physical basis of Chi’s regulation of RecBCD is, however, unknown. Beginning at a double-strand (ds) end in broken DNA, RecBCD rapidly unwinds DNA with its fast RecD helicase moving on the 5'-ended strand and slower RecB helicase moving on the 3'-ended strand (Taylor and Smith 2003). A single-stranded (ss) loop thus accumulates on the 3'-ended strand and grows as the reaction proceeds (Fig. 1A,B; see also Fig. 3A, below).
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); subsequently, the three subunits disassemble, perhaps at the end of the DNA, and the enzyme remains inactive (Taylor and Smith 1999). In reactions with excess Mg2+ ions over ATP, RecBCD’s exonuclease activity is high, and the enzyme degrades the 3'-ended strand up to Chi (Dixon and Kowalczykowski 1993), nicks the complementary strand (Taylor and Smith 1995b), and then degrades the 5'-ended strand during continued unwinding (Anderson and Kowalczykowski 1997a). At least under this condition, the enzyme begins to load RecA protein onto the 3'-ended strand to the left ("downstream") of Chi (Anderson and Kowalczykowski 1997b), and the single-stranded DNA (ssDNA)-RecA filament undergoes strand exchange with a homologous duplex (Dixon and Kowalczykowski 1991). This joint molecule has been postulated to form recombinants by a break-copy scheme (break-induced replication) or by formation and resolution of a Holliday junction (Fig. 1B; Smith 1991).
Crucial to the production of recombinants is the alteration of RecBCD’s activities at and by Chi. The stimulation of recombination by Chi can be up to 30-fold (Stahl and Stahl 1977; Schultz et al. 1983), and recBCD mutants specifically lacking the ability to respond to Chi have reduced recombination proficiency (Schultz et al. 1983; Lundblad et al. 1984). Two classes of mutants lacking Chi hot spot activity have mutations in recC (see Discussion). The amino acids altered in these mutants (Arnold et al. 2000; S.K. Amundsen, unpubl.) line part of a tunnel in the structure of RecBCD cocrystallized with hairpin DNA (Fig. 1C,D). It has been postulated that RecC recognizes Chi as the 3'-ended strand moves from the RecB helicase domain through the tunnel in RecC on its way to the nuclease domain of RecB (Singleton et al. 2004). The steps between Chi recognition and alteration of the nuclease and RecA loading activities are unknown. We describe here a novel class of recB mutant enzymes whose properties indicate that the RecD subunit signals the RecB subunit to cut DNA. These observations lead us to propose a new hypothesis for the regulation of wild-type RecBCD by Chi: a cascade of intersubunit signals from Chi–RecC to RecD to RecB.
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Results |
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Previous studies of recBCD mutants that lack some but not all RecBCD activities have helped to elucidate how Chi regulates RecBCD enzyme (e.g., Schultz et al. 1983; Lundblad et al. 1984; Amundsen et al. 1990, 2002; Yu et al. 1998b; Amundsen and Smith 2007). To find additional novel mutants, we targeted mutations in DNA encoding the C-terminal 381 amino acids, residues 800–1180, of RecB. This region contains the nuclease and RecA loading domains (Yu et al. 1998b; Spies and Kowalczykowski 2006), two activities altered by Chi. Using a mutagenic PCR and colony-screening procedure, we found 11 isolates that were recombination deficient (Rec–) in Hfr crosses but retained RecBCD exonuclease activity (Nuc+) as indicated by resistance to phage infections (see below; Schultz et al. 1983) or by assay of cell-free extracts (S.K. Amundsen, unpubl.). Each isolate contained two to 10 missense mutations, or 57 mutations in all. Twelve of these mutations were clustered in codons 800–810, of which five were in codon Y803 and two in codon V804. For further analysis, we made single codon mutations, each of which was among the initial 57 mutations, to create two new alleles: recB2732 (Y803H) and recB2734 (V804E). These altered amino acids are in the conserved helicase motif VI of RecB (Fig. 1D; see Discussion). The cellular phenotypes and enzymatic activities in extracts of these mutants were similar to those of the original isolates containing the corresponding mutations. The data presented here were obtained with the single codon mutations.
The two new mutants were nearly as Rec– as strains with a 
recBCD-null allele. In Hfr crosses, the recombination proficiency of V804E was reduced by a factor of 
500, like recBCD, and that of Y803H by a factor of 200 (Table 1). In phage 
crosses, in which recombination is less dependent on RecBCD (Stahl and Stahl 1977), the proficiencies were reduced by a factor of 7, similar to that of the recBCD null. In these crosses, we measured Chi hot spot activity, the ratio of the recombinant frequency in an interval with Chi to that in the same interval without Chi (Stahl and Stahl 1977). recBCD+ cells gave a Chi activity of 5.3, whereas a recBCD-null mutant gave no Chi activity (ratio of 1), as reported previously (Stahl and Stahl 1977). Like recBCD-null mutants, the new mutants lacked detectable Chi activity (i.e., they were Rec– Chi–) (Table 1).
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). T4 gene 2 mutant phage formed plaques with the same low efficiency (10–6) on the new mutants as on recBCD+ cells (Table 1) but formed plaques with near unit efficiency on previously isolated mutants lacking RecBCD exonuclease (recBCD or recD).
We next tested the exonuclease activity of RecBCD enzymes purified from the mutants. As expected from the resistance of the mutants to T4 gene 2 mutant phage (Table 1), the mutant enzymes had nearly wild-type levels of ATP-dependent ds exonuclease activity (Table 2), the hallmark of RecBCD enzyme (Smith 1990). We noted, however, that at very low ATP concentration (25 µM), the enzymes had little ds exonuclease activity (Supplementary Fig. S1). Half maximal ds exonuclease activity required 0.5–2 mM ATP (Supplementary Fig. S1), closer to the intracellular ATP concentration of 3 mM than the standard assay concentration of 25 µM (Eichler and Lehman 1977). The ATP-dependent ssDNA exonuclease activity of the mutant enzymes, compared with that of the wild-type enzyme, was indistinguishable at low ATP concentration (A.F. Taylor, unpubl.) and similar at high ATP concentration (Table 2). Thus, these mutants are Rec– Nuc+ Chi– and may have alterations in Chi’s regulation of RecBCD enzyme.
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Wild-type RecBCD enzyme cuts DNA a few nucleotides to the 3' side of the Chi sequence 5'-GCTGGTGG-3' (Taylor et al. 1985). When we tested the mutant enzymes for this activity, we observed that they failed to cut at Chi (Fig. 2A), as expected from the lack of Chi hot spot activity in the mutants (Table 1). Instead, each mutant enzyme cut at a novel position, different for each mutant enzyme (Fig. 2A). Remarkably, these novel positions depended on the length of the DNA substrate. Eight substrates, each with the same 5'-labeled end and ranging in length from 1.1 kb to 4.4 kb, were made and reacted with each enzyme. The lengths of the novel 5'-labeled products were determined by gel electrophoresis (Supplementary Fig. S2).
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Ratio of RecB:RecD helicase rates strictly correlates with the position of DNA cutting
As it unwinds DNA, wild-type RecBCD forms two ssDNA tails and a ssDNA loop that moves along the DNA and grows as the reaction proceeds (Fig. 1A; Taylor and Smith 1980), a consequence of RecB moving more slowly on the 3'-ended strand than RecD moves on the 5'-ended strand (Taylor and Smith 2003). The ratio of the lengths of the short and long tails is the ratio of the rates of RecB and RecD movement on their respective strands (Fig. 1A, x/y). To determine if these rates were altered in the recB mutants, we examined by electron microscopy (EM) DNA molecules partially unwound by the wild-type or mutant enzymes (Fig. 3). The rate of elongation of the long tail was not significantly different in the mutants or wild type, indicating that the RecD helicase rate was not detectably altered in the mutants (Table 2). The rate of elongation of the short tail by the mutants was, however, markedly less than that by the wild type, indicating that the mutant RecB helicases moved at only 39% (Y803H) or 31% (V804E) of the rate of wild-type RecB. The ratio of the rates of elongation of the short and long tails (i.e., the RecB:RecD helicase rates, x/y), was 0.28 for Y803H and 0.19 for V804E, compared with 0.56 for wild-type RecBCD.
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). Under reaction conditions identical to those used in the EM analysis, with 1 mM Mg2+, the position of the cuts, as a fraction of the lengths of the DNA substrates, was 0.26 for Y803H and 0.18 for V804E (Table 2; Supplementary Fig. S3). For each mutant enzyme, this position was indistinguishable from the independently determined ratio of the RecB:RecD helicase rates (x/y), 0.28 for Y803H and 0.19 for V804E (Table 2). From the two sets of data above, we conclude that the mutant enzymes cut the DNA when RecD reaches the end of the substrate strand on which it travels. At that moment, RecB, with the nuclease domain, would be a fraction of the distance along the other strand, determined by the ratio x/y, and would cut at that position (Fig. 1A). With the observations below, we infer that in these mutants, when RecD stops unwinding, it signals RecB to cut the DNA.
Collision of RecBCD enzymes on DNA changes the position of the novel cuts
When two wild-type RecBCD enzyme molecules act on one DNA substrate, with one enzyme entering from each end, the enzymes collide near the middle of the molecule and cut the DNA there (Dixon and Kowalczykowski 1993). This situation occurs most often at high [RecBCD] relative to [DNA]. We used such collisions to stop the mutant RecBCD enzymes half-way along the DNA. As reported previously, we observed that wild-type RecBCD frequently cut near the middle of the DNA substrate at high concentration, when two enzyme molecules can come from each end, but infrequently at low concentration, when only one enzyme molecule is present on the DNA (Fig. 4). Instead, at low concentration, wild-type RecBCD cut at Chi, which was located beyond the mid-point as RecBCD approached Chi in the active orientation.
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), we infer that termination of RecD’s travel induces RecD to signal cutting by the RecB subunit. (It is not clear why wild-type RecBCD at high concentration cuts in the middle of the substrate rather than at one-half of its characteristic x/y ratio of RecB:RecD helicase rates. Failure to cut there may be related to its failure to cut at low concentration at any point in the absence of Chi [Fig. 2A].)
Slowing RecD helicase abolishes cutting at novel positions and can revive Chi cutting
To test more directly the hypothesis that the mutant enzymes cut DNA when RecD translocation terminates at the end of its substrate strand, we coupled the recD2177 (K177Q) mutation with the new recB2732 (Y803H) or recB2734 (V804E) mutation to make doubly mutant RecBCD enzymes. The recD2177 (K177Q) mutation, in the ATPase site, slows RecD to 32 nucleotides (nt) per second, 5% of the wild-type rate (Taylor and Smith 2003). For the Y803H and Y804E singly mutant enzymes, the rates of RecB-mediated unwinding are 122 and 97 base pairs (bp) per second, respectively (Table 2). Consequently, in the doubly mutant enzymes, RecB is expected to move more rapidly than RecD; RecB would reach the end of the substrate before RecD, and the signal to cut the DNA might not be generated. As predicted, the novel length-dependent cuts made by the single recB mutant enzymes were not detectably made by the doubly mutant enzymes (Fig. 2A; Supplementary Fig. S4). Less than 2% of the DNA was cut by the doubly mutant enzymes, whereas 35% of the DNA was cut by the singly mutant enzymes. These results indicate that the RecD subunit is involved in generating the novel length- dependent cuts on DNA.
Remarkably, altering the RecD subunit, in the RecBY803HCDK177Q enzyme, restored Chi-dependent cuts (Fig. 2A). This is consistent with cells bearing this doubly mutant enzyme showing Chi hot spot activity (Table 1) and with RecBY803H and likely RecC encountering Chi before the very slow RecDK177Q subunit gets to the end of the DNA (Table 2). The RecBV804ECDK177Q enzyme did not cut at Chi (Supplementary Fig. S4), consistent with the lack of Chi hot spot activity in cells with this doubly mutant enzyme (Table 1).
Mutant enzymes do not load RecA onto the novel cut products
After encountering Chi, wild-type RecBCD loads RecA protein onto the newly generated 3' end (Anderson and Kowalczykowski 1997b). To determine if this Chi-dependent alteration occurs in the mutant enzymes after they generate their length-dependent cuts, we tested RecA loading onto DNA substrates with or without Chi. Each mutant RecBCD enzyme made the expected novel cut fragments, but in neither case was there detectable loading of RecA onto this product (Supplementary Fig. S5). RecA loading was measured by resistance of the cut product to digestion by exonuclease I, which is specific for 3' ssDNA ends and is inhibited by RecA protein on the DNA. The failure to load RecA can account for the Rec– phenotype of these mutants (Table 1), the basis of their isolation.
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Discussion |
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Amino acid substitutions in helicase motif VI slow RecB and confer a novel DNA cutting activity dependent on the substrate length
The two mutants described here change two highly conserved amino acids in helicase motif VI, whose consensus sequence in 39 bacterial RecB proteins is RLLYVA-TR, where "-" is a not-well-conserved amino acid. In E. coli’s RecB, this sequence is RLLYVALTR; the mutants described here are altered in the amino acids underlined. In RecBCD this sequence is part of a 24-amino-acid-long 
-helix that does not appear to contact DNA during unwinding but lies close and parallel to another short helix that likely does (Singleton et al. 2004). Studies of mutants with alterations in motif VI in several superfamily I helicases suggest that amino acids in this motif are required to couple ATP hydrolysis to DNA movement (e.g., Graves-Woodward et al. 1997). In one case, E. coli UvrD (helicase II), the T 
A amino acid alteration in this motif changes the conformation of the protein, as indicated by increased sensitivity to limited proteolysis (Hall et al. 1998). Thus, this long -helix may be important in transducing information, via a conformational change, between the ATPase and helicase active sites. This interpretation is consistent with the increased apparent KM for ATP and the decreased RecB helicase rates in the RecBCD mutants studied here (Table 2; Supplementary Fig. S1). We suggest that in wild-type RecBCD, this helix is important also for the transduction of the Chi-dependent signal that alters the activities of the enzyme after acting at Chi (Taylor and Smith 1992).
The mutations studied here impart a novel phenotype to RecBCD—the ability to determine a fraction of the length of the DNA substrate and to cut the DNA at that point. The simplest interpretation is that this "calculation" reflects the ratio of the rates of movement of the RecB and RecD helicases (Fig. 1A, x/y; Table 2). In the mutants, the RecD helicase moved at the same rate as it does in wild-type RecBCD, but the RecB helicase was slower than that in wild type, 31% of the wild-type rate for V804E and 39% for Y803H. The near equality of the RecB:RecD ratio and the position of the cut for each enzyme (Table 2) supports this simple interpretation. Furthermore, since RecB has the nuclease domain (Yu et al. 1998b) and the position of the cut is indistinguishable from the point at which RecB’s nuclease domain would be when RecD reaches the end of the DNA, we conclude that cutting, by RecB, is induced when RecD reaches the end of the DNA and stops unwinding DNA.
We tested the interpretation that the mutant enzymes cut DNA when RecD stops unwinding in two ways. (1) Introduction of the recD2177 (K177Q) mutation in the RecD ATP site (Korangy and Julin 1992) slows the RecD helicase to 5% of the wild-type RecD rate (Taylor and Smith 2003). This rate is slower than that of the RecBY803H or RecBV804E mutant subunit (Table 2). Thus, in the doubly mutant enzymes RecB is expected to reach the DNA end before RecD does. As predicted, these doubly mutant enzymes did not cut at the novel length-dependent position (Fig. 2A; Supplementary Fig. S4). (2) When two wild-type RecBCD molecules simultaneously unwind a DNA molecule, one from each end of the DNA, the enzymes cut when they collide near the middle of the DNA (Dixon and Kowalczykowski 1993). When the new mutant enzymes were similarly tested, cutting occurred at approximately one-half the distance from the entry end to the position of the novel cut observed at low enzyme concentration, when only one enzyme molecule is present on the DNA (Fig. 4). Thus, whether RecD stops at the end of the DNA or upon collision with another RecBCD molecule, cutting is induced at the position where RecB is expected to be located.
Wild-type RecBCD generates a 3' DNA end a few nucleotides 3' of the Chi sequence. With excess ATP, this occurs by a simple nick (Taylor et al. 1985), whereas with excess Mg2+, degradation of the 3'-ended strand ceases at or near Chi (Dixon and Kowalczykowski 1993; Taylor and Smith 1995b). The mutants studied here appear to generate 3' ends in a similar manner under these two reaction conditions (Fig. 2; Supplementary Fig. S6; S.K. Amundsen and A.F. Taylor, unpubl.) but at novel length-dependent positions. We suppose that the basic mechanism that induces the cut is the same as that in wild-type RecBCD but that the signal for this induction is different, as discussed below. Although new 3' ssDNA ends were produced by both the mutant and wild-type enzymes, the mutants were recombination deficient (Table 1). This deficiency is likely due to the mutants’ inability to load RecA protein onto the newly generated 3' end (Supplementary Fig. S5). Thus, the mutants mimic only part of the change at Chi—they cut DNA but do not load RecA.
Our results indicate that in the mutant enzymes, RecD signals RecB to cut the DNA. Below, we extend this explanation into a new hypothesis for how, in wild-type enzyme, Chi signals RecB to cut the DNA and to load RecA protein to initiate strand exchange.
A ‘signal transduction cascade’ hypothesis for Chi’s regulation of wild-type RecBCD enzyme
Based on these and other observations, especially the RecBCD crystal structure and its interpretation (Singleton et al. 2004), we propose the following hypothesis for Chi’s regulation of wild-type RecBCD enzyme (Fig. 5). The 3'-ended strand of DNA passes through the RecB helicase and into a tunnel in RecC (Fig. 1C). Critical amino acids in RecC engage Chi (5'-GCTGGTGG-3') on that strand, which is necessary and sufficient for maximal Chi activity (Smith et al. 1981; Bianco and Kowalczykowski 1997), and RecC signals the RecD helicase to stop unwinding DNA. When RecD stops, it signals RecB to cut the DNA four to six nucleotides to the 3' side of Chi (Taylor et al. 1985) and to begin loading RecA onto the newly generated 3'-ended ssDNA (Anderson and Kowalczykowski 1997b). Unwinding continues, with RecB now the leading helicase and loading RecA at intermittent points. RecA’s cooperative binding fills the gaps to form a continuous RecA-ssDNA filament, which undergoes strand exchange with a homologous duplex (Fig. 1B). At an as-yet-undetermined point, RecBCD dissociates from the DNA, and the three subunits disassemble, leaving RecBCD inactive (Taylor and Smith 1999).
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0.1 nM) and unwinds a few base pairs, with the 3' end contacting RecB and the 5' end contacting RecC and RecD (Fig. 1; Ganesan and Smith 1993; Taylor and Smith 1995a; Farah and Smith 1997). An open tunnel in RecB and RecC is plausibly the path of the 3'-ended strand toward the nuclease domain in RecB (Singleton et al. 2004). Alteration of five to eight amino acids in RecC in a class of compensating frameshift mutations, called recC* (Schultz et al. 1983; Arnold et al. 2000) or of a single amino acid in the recC343 (TexA) mutant (Lundblad et al. 1984; S.K. Amundsen, unpubl.) greatly reduces Chi activity. These amino acids line part of the tunnel in RecC. The distance from these amino acids to the nuclease active site would readily accommodate four to six nucleotides (Singleton et al. 2004), the distance between Chi and the cuts that depend on it (Taylor et al. 1985).
Consistent with our hypothesis, RecD plays a central role in the regulation of RecBCD. Although the nuclease active site is in RecB (Yu et al. 1998b; Zhang and Julin 1999), recD-null mutants are nuclease deficient (Amundsen et al. 1986). RecD must therefore regulate RecB’s nuclease activity. In addition, RecBC, but not RecBCD, loads RecA protein in the absence of Chi (Churchill et al. 1999); RecD must therefore inhibit the loading of RecA, which may be directed by part of RecB (Spies and Kowalczykowski 2006). recB mutants altered in the nuclease active site are Rec– and do not load RecA unless the RecD subunit is removed (Anderson et al. 1999; Amundsen et al. 2000). It is therefore plausible that the Chi signal, which alters the nuclease and RecA loading activities, is transmitted via RecD. In the crystal structure, the ordered part of RecD does not contact RecB (Fig. 1C; Singleton et al. 2004) but does come within 0.8 nm of the RecB nuclease domain, which may overlap the RecA loading domain (Spies and Kowalczykowski 2006). We propose that Chi alters the conformation of RecC, which does contact RecD, and moves RecD against the RecB nuclease domain, thereby altering its activity. Alternatively, RecD may contact RecB during unwinding and, after receiving the Chi signal from RecC, alter the RecB helicase domain, which in turn affects the RecB nuclease domain via the 30-amino-acid-long tether connecting these domains (Fig. 1C); this possibility is consistent with our interpretation of the recB helicase domain mutants described in this study.
Studies of single RecBCD molecules by fluorescence microscopy also support this hypothesis. Unwinding of the DNA duplex pauses at or near Chi, and the length of the pause is proportional to the distance of the Chi site from the DNA end at which RecBCD initiated unwinding (Spies et al. 2003). We interpret these results to mean that RecD, the leading helicase before Chi (Taylor and Smith 2003), stops unwinding at or near Chi, in accord with our hypothesis (Fig. 5). RecB continues, perhaps without any pause, to travel along the ss loop for a time proportional to the loop length (i.e., also proportional to the distance of Chi from the DNA end) (Fig. 1A). After traversing the loop, RecB becomes the leading (unwinding) helicase after Chi but at a rate slower than RecD was before Chi, as observed (Spies et al. 2003). We suppose that the slowing or elimination of the RecD helicase reflects a conformational change as part of the Chi signal transduction.
Our hypothesis is distinct from previous hypotheses of how Chi affects RecBCD enzyme. According to one hypothesis (Thaler et al. 1988), RecD is ejected at Chi. Early enzymatic studies argued against this possibility, however: After acting at Chi, the enzyme retains nuclease activity (Taylor and Smith 1992), whereas RecBC (i.e., without RecD) lacks nuclease activity (Amundsen et al. 1986). Furthermore, subsequent light microscopy studies of single RecBCD molecules showed that RecD remains with the enzyme after Chi (Dohoney and Gelles 2001; Handa et al. 2005). According to another hypothesis (Yu et al. 1998a), the RecB nuclease domain "swings" from one side of the enzyme, where it digests the 3' 5' strand, to the other side, where it digests the 5' 3' strand. Singelton et al. (2004) modified this view and hypothesized that at Chi the 3' 5' strand, the one with Chi, moves from a channel in RecC aimed toward the nuclease site into another channel aimed away from the nuclease site. This change might be effected by a RecB -helix swinging to block the first channel. This hypothesis is consistent with ours, which in addition specifies how Chi effects this and other changes in the enzyme.
Although aspects of our hypothesis (Fig. 5) are speculative and require further support, it is consistent with current observations, as noted above, and makes testable predictions. Specific mutations in each gene should disrupt the signal transduction cascade. In addition to the recC* mutations that appear to abolish the Chi–RecC interaction (see above), there should be mutant forms of RecC that cannot transmit the signal to RecD, mutant forms of RecD that cannot receive the signal from RecC or that cannot transmit the signal to RecB, and mutant forms of RecB that cannot receive the signal from RecD. Previously described recC and recB mutations may correspond to these classes: recC2145, recB2154, and recB2155 are Rec– Nuc+ Chi–, like the mutants described here, and may be unable to transmit or receive the Chi signal (Amundsen et al. 1990). Our model predicts a class of recD mutations that cannot receive or transmit the signal and would be Rec–. Such mutations have not been reported to date, but recD-null mutations are Rec+ Nuc– Chi–, as predicted if Chi annuls the regulatory roles of RecD (Amundsen et al. 2000). We suppose that the transduction of the Chi signal involves conformational changes in each of the RecBCD subunits; such changes might be detectable by limited proteolysis or spectroscopy of fluorescently labeled subunits.
Intersubunit signaling in other complex protein machines
The conceptual model of intersubunit signaling, such as that proposed here for RecBCD, may be applicable to a broad range of complex protein machines. Particularly relevant here are two examples of enzymes with three types of subunits and multiple activities on DNA, like RecBCD. (1) Mismatch correction in E. coli depends on the MutS, MutH, and MutL proteins. MutS binds to mismatched bases in DNA, the MutH latent endonuclease binds to a distant hemimethylated DNA site, and MutL appears to connect MutS and MutH (Iyer et al. 2006). The MutH nuclease is activated by MutS and MutL in the presence of ATP and a mismatch. The mechanism of the activation is unclear but likely involves transduction of a signal from MutS to MutH, perhaps via MutL. (2) Type I restriction enzymes bind to a specific DNA sequence, travel along the DNA, and cut at a distant site when travel stops, due to collision between two enzymes or a structural constraint in the DNA (Murray 2000). In these enzymes, the HsdS subunit binds a specific DNA sequence but is aided by the HsdM subunit, which contains the methyltransferase domain; the HsdR subunit contains the endonuclease domain. If the DNA sequence is hemimethylated, the HsdM subunit acts before travel is initiated, but if the sequence is unmethylated, travel commences and the HsdR subunit acts. Signaling between the subunits therefore must regulate modification versus restriction. Another related example is the type II DNA topoisomerases, in which there appears to be signaling between the ATPase site in one subunit and the DNA breaking–rejoining "gate" in another subunit (Bates and Maxwell 2007). Mutational alterations of these proteins may help elucidate the putative intersubunit signal transduction, as reported here for RecBCD enzyme.
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Materials and methods |
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Bacterial strains are listed in Supplementary Table S1 with their genotypes and sources. Plasmids are listed in Supplementary Table S2. For visual clarity, allele numbers and polypeptide designations are expressed as superscripts when more than one recBCD gene or RecBCD polypeptide are designated. Bacterial strains were constructed by phage P1 transduction, CaCl2-mediated transformation, electroporation, or "recombineering" (Ausubel et al. 2003; Thomason et al. 2005). Plasmids were constructed by standard procedures (Ausubel et al. 2003).
Culture media and genetic assays
Culture media have been described (Cheng and Smith 1989). Chi hot spot activity and recombination proficiency were measured in vegetative crosses (Stahl and Stahl 1977), and E. coli recombination proficiency in Hfr crosses (Schultz et al. 1983).
Mutant isolation
Mutations in the C-terminal part of recB were generated by mutagenic PCR (Ausubel et al. 2003). Two primer pairs were used—one amplifying codons 712–1181, and the other pair codons 794–1181 (Supplementary Table S3); recB has 1181 codons including that for termination. Each PCR contained, in 100 µL, 20 fmol of plasmid pDWS2 DNA, 30 pmol of each primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.5 mM MnCl2, 0.01% gelatin, 1 mM dCTP and dTTP, 0.2 mM dATP and dGTP, and 5 U of Taq polymerase (Boehringer Mannheim Biochemicals) and ran for 30 cycles (1 min at 94°C, 1 min at 50°C, and 1 min at 72°C). The PCR products were digested with BglII, which cleaves between recB codons 797 and 798, and with BseRI, which cleaves four nucleotides after the recB termination codon. The largest fragment (1.1 kb) was "swapped" with the corresponding recB+ fragment of pMR3. Approximately 5000 ampicillin-resistant (AmpR) transformants of strain V2959 [recBCD2731::kan (lacIZYA–argF)U169] were screened for recombination deficiency (Rec–) by toothpick transfer of colonies to minimal lactose agar plates containing ampicillin (100 µg/mL) and kanamycin (25 µg/mL) and spread with 108 stationary-phase cells of strain KL226 (lac+ Hfr PO 12.2). Approximately 1000 Rec– colonies (those unable to generate Lac+ AmpR KanR recombinant colonies) were tested by cross-streaking colonies from an LB-ampicillin master plate onto LB agar with phage P2 (applied as lines of 20 µL of 108 phage per milliliter); growth of P2 appears to require RecBCD exonuclease (Nuc+) (Amundsen et al. 1990). (Many of the Rec– Nuc– isolates had plasmids without the BglII–BseRI fragment, reflecting inefficient swapping.) Lawns of 200 Rec– Nuc+ candidates were tested by spot tests for growth of (red–gam) 
+ and o phages, which do not make plaques on Rec– Nuc+ strains, and of phage P1, which does not make plaques on Rec– strains (Schultz et al. 1983; Amundsen et al. 1990, 2000). Plasmids from 50 stable Rec– Nuc+ candidates were isolated and introduced into strain V2831 (recBCD2731::kan); these transformants were tested quantitatively as in Table 1. The recB nucleotide sequence was determined for 11 of the most Rec– candidates. The recB2732 (Y803H) and recB2734 (V804E) mutations were introduced into plasmid pSA124 using the QuikChange kit (Stratagene) and mutant oligonucleotides (Supplementary Table S3). These mutations were transferred to plasmid pMR3 by fragment swapping as described above. Double recB recD mutants were constructed by site-directed mutagenesis of pMR3 to introduce the recD2177 mutation, followed by swapping of the BseRI–BglII fragment from pSA176 or pSA178 to introduce the recB2732 or recB2734 mutation.
Enzyme purification and assays
The purification of wild-type RecBCD enzyme has been described (Taylor and Smith 2003). Mutant RecBCD enzymes were purified from strain V2831 (recBCD2731) containing derivatives of pMR3 by similar methods. In brief, cells from a 12 L of culture in Terrific Broth (Fisher) were lysed, and enzymes were purified by column chromatography—HiTrap Q Sepharose, HiPrep Sephacryl S-300 HR, and HiTrap Heparin (all from GE Lifesciences), followed by CHTII hydroxyapatite (Bio-Rad) for the single mutants or ssDNA agarose (GE Lifesciences) for the double mutants. The final product (1 mg) was judged to be 80% pure by staining with SimplyBlue (Invitrogen) an SDS–polyacrylamide gel loaded with 0.5 µg of protein.
Assays for RecBCD ds and ss exonuclease used native and boiled [3H] T7 DNA, respectively (Eichler and Lehman 1977). Gel-electrophoretic assays for DNA unwinding, Chi cutting, and RecA loading were as described (Taylor et al. 1985; Amundsen et al. 2000; Taylor and Smith 2003) with 5 mM ATP, 3 mM Mg(OAc)2, and 1 µM SSB (Promega), except as noted in Figure 4 and Supplementary Figures S3 and S5. Agarose gels (0.7%; 22 cm long) in TBE buffer (Ausubel et al. 2003) were run at room temperature for 2.5 h at 100 V (Fig. 2A; Supplementary Figs. S4, S5) or for 16 h at 50 V (Figs. 2B, 4; Supplementary Figs. S2, S3). Analysis of Typhoon Trio PhosphorImage files (GE Lifesciences) used ImageQuant TL software (Amersham); size markers were fit to a log-linear straight line (r2 > 0.997). EM assays for DNA unwinding (Taylor and Smith 2003) contained 5 mM ATP, 1 mM Mg2+, and 1 µM SSB; rates were calculated from molecules whose complementary unwound strands differed in length by <33%.
For the experiments in Figures 2A and 4 and Supplementary Figures S4 and S5, DNA substrates were prepared from pBR322 +F or o (4361 bp) by cutting with HindIII, treating with phosphatase, labeling the 5' ends using polynucleotide kinase and [
-32P] ATP (Amundsen et al. 2000), and cutting with ClaI, which produces a 4355-bp fragment with one of the two 32P labels and two short fragments (five and seven nucleotides) not seen in our analyses. For the experiments in Figure 2B and Supplementary Figures S2 and S3, the DNA substrates were similarly prepared from pBR322 o DNA by cutting with StyI, labeling the 5' ends, cutting with BsmI, and separating the 4351-bp fragment from a short fragment using an S200 spin column. Subfragments for RecBCD reactions and for size markers were produced by subsequent digestion of the 4351-bp fragment with NruI, SalI, HindIII, PvuI, AlwNI, AflIII, NdeI, or Tth111I.
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2 Present address: Center for Cellular and Molecular Biology, Hyderabad 500 007, India.
E-MAIL gsmith{at}fhcrc.org; FAX (206) 667-6497.
Supplemental material is available at http://www.genesdev.org.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1605807
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