Chi hotspot Control of RecBCD Helicase-nuclease: Enzymatic Tests Support the Intramolecular Signal-transduction Model

Abstract

Repair of broken DNA is essential for life; the reactions involved can also promote genetic recombination to aid evolution. In Escherichia coli, RecBCD enzyme is required for the major pathway of these events. RecBCD is a complex ATP-dependent DNA helicase with nuclease activity controlled by Chi recombination hotspots (5'-GCTGGTGG-3'). During rapid DNA unwinding, when Chi is in a RecC tunnel, RecB nuclease nicks DNA at Chi. Here, we test our signal transduction model - upon binding Chi (step 1), RecC signals RecD helicase to stop unwinding (step 2); RecD then signals RecB (step 3) to nick at Chi (step 4) and to begin loading RecA DNA strand-exchange protein (step 5). We discovered that ATP-γ-S, like the small molecule RecBCD inhibitor NSAC1003, causes RecBCD to nick DNA, independent of Chi, at novel positions determined by the DNA substrate length. Two RecB ATPase-site mutants nick at novel positions determined by their RecB:RecD helicase rate ratios. In each case, we find that nicking at the novel position requires steps 3 and 4 but not step 1 or 2, as shown by mutants altered at the intersubunit contacts specific for each step; nicking also requires RecD helicase and RecB nuclease activities. Thus, altering the RecB ATPase site, by small molecules or mutation, sensitizes RecD to signal RecB to nick DNA (steps 4 and 3, respecitvely) without the signal from RecC or Chi (steps 1 and 2). These new, enzymatic results strongly support the signal transduction model and provide a paradigm for studying other complex enzymes.

Keywords: Chi recombination hotspots; DNA break repair and genetic recombination; Intramolecular signal transduction mutants; RecBCD helicase-nuclease; Small molecule inhibitors.

Figure 1.. Models for RecBCD structure, RecBCD-promoted recombination, and RecBCD’s control by Chi recombination hotspots.

(A) A cryoEM structure (PDB 6SJB) of RecBCD bound to hairpin-shaped DNA with long 5’ and 3’ ss tails, the latter containing Chi [10]. RecB (orange), RecC (blue), and RecD (green) have the indicated functional domains. Four amino acids (two are shown in red) of the RecB nuclease domain have charges opposite to those of four amino acids (yellow) on the RecC loop (cyan); each set is important for Chi hotspot activity and can be computationally fit to the other by swinging the nuclease domain on the RecB tether (magenta line delimited by arrows) [26]. (B) Model for genetic recombination promoted by RecBCD enzyme. RecBCD binds a dsDNA end (a) and unwinds the DNA, producing a loop and two tails (b) which can anneal to produce two growing loops (c). Upon encountering Chi (5’-GCTGGTGG-3’) on the top strand, RecBCD nicks that strand (d) and loads RecA onto it (e). The ssDNA-RecA filament invades intact homologous DNA, forming a D-loop (f), which can be converted into a Holliday junction (g) to produce reciprocal recombinants; alternatively, DNA synthesis (h) primed in the D-loop can lead to non-reciprocal recombinants. From [23]. (C) Signal transduction model for Chi’s control of RecBCD. When Chi is recognized in the RecC tunnel (step 1), RecC signals RecD to stop unwinding (step 2). When stopped, RecD signals RecB to swing Nuc (step 3), cleave the DNA (step 4) and begin loading RecA (step 5) (see Fig. 1B). Modified from [23]. (D) RecB nuclease domain (Nuc) swing model. Without DNA, RecBCD has the conformation of that in a crystal structure (PDB 1W36) [8] with Nuc at the RecC tunnel exit. Upon DNA binding, Nuc (grey) swings to the RecC loop; upon Chi’s encounter, Nuc swings back to the RecC tunnel exit and nicks DNA at Chi. A further change, such as Nuc rotation, allows it to load RecA. From [20].

Figure 2.. Small molecules ATPγS and NSAC1003 convert RecBCD to a Chi-activated state dependent on signal transduction steps 3 and 4 but not 1 or 2.

(A) ATPγS and NSAC1003 induce RecBCD to cleave DNA at a hotspot independent of Chi. pBR322 χ° DNA (0.4 nM) linearized with HindIII, labeled at the 5’ ends, and cleaved with ClaI (six bp from the right-end HindIII site) was reacted with purified RecBCD enzyme (0.3 or 0.11 nM) as described in Materials and Methods. Reactions contained ATP (5 mM except 2.5 mM with ATPγS), NSAC1003 (200 μM), or ATPγS (2.5 mM) or combinations as indicated. Reaction products were analyzed by electrophoresis in a 0.9% agarose gel and autoradiographed. DS, double-stranded DNA; SS, single-stranded DNA produced by boiling (+);✖: NSAC1003-induced cleavage product; ✦: ATPγS-induced cleavage product; M, single-stranded DNA markers with the indicated nucleotide lengths. (B) ATPγS-induced nicking occurs on χ° or χ⁺E224 DNA. Reactions were as described in (A). (C) Intramolecular signaling steps 3 and 4, but not 1 or 2, are required to respond to ATPγS. Extracts from RecBCD wild-type and mutants (0.5 or 0.25 mg of extract protein/ml of reaction mix) deficient in each step of the signal transduction pathway (Table 1) were assayed on pBR322 χ° DNA as described in (A). (D) As in panel C but with NSAC1003 (200 μM) in place of ATPγS and 0.33 mg of extract protein/ml of reaction mix. Mutants were, for step 1, RecBC^S39VD; step 2, RecBC^Δ541–544D^Δ97–99; step 3, RecB^{[634–635, 639, 643–644, 646]Ala}CD^Δ523–526; and step 4, RecB^Δ913–922 C^Δ599–608D (Table 1).

Figure 3.. Position of the ATPγS-dependent cut depends on DNA substrate length and ATPγS concentration.

(A) pBR322 χ⁺E224 DNA (0.4 nM) was digested with HindIII, labeled on the 5’ ends, and further digested with ClaI (substrate 1), NdeI (substrate 2) or StyI (substrate 3) to produce substrates 4354 bp, 2270 bp or 1350 bp long, respectively, with Chi 962 bp from the labeled end. Reactions were as described in Fig. 2A with 1 nM or 0.3 nM RecBCD enzyme. (B) pBR322 χ⁺E224 DNA (0.4 nM), labeled on the 5’-ends and cut with ClaI, was reacted with 0.3 nM RecBCD enzyme and the indicated concentrations of ATP and ATPγS. Products were analyzed as in Fig. 2A. ✦ and Chi (χ⁺E): ATPγS-induced and Chi-dependent cleavage products, respectively.

Figure 4.. RecB ATPase site mutants respond to ATPγS and require intramolecular signal transduction step 3, but not 2, to make DNA length-dependent cuts.

(A) Purified RecBCD enzyme (0.3 or 0.1 nM; wt or the indicated mutant) was assayed with pBR322 χ⁺F225 DNA (0.4 nM) and analyzed as in Fig. 2A. (B) Extracts (0.5 or 0.17 mg of extract protein/ml of reaction mix) from RecB^Y803HCD and RecB^V804ECD mutants without (wt) or with additional mutations blocking step 2 or step 3 of the signal transduction pathway (Fig. 1C; Table 1) were assayed on pBR322 χ° DNA and analyzed as in Fig. 2C without ATPγS. ✦, ◈, and Chi (χ⁺F): ATPγS-induced, ATPase-site mutant-dependent, and Chi-dependent cleavage products, respectively. See also Fig. S5.

Figure 5.. ATPγS- and NSAC1003-dependent cuts require the RecB and RecD ATPases.

Purified RecBCD (0.1 or 0.3 nM), RecB^K29QCD (1 nM or 0.33 nM), or RecBCD^K177Q (1 nM or 0.33 nM) was assayed with pBR322 χ⁺E224 DNA (0.4 nM) and analyzed as in Fig. 2A. (A) Reactions contained either 5 mM ATP or 2.5 mM ATP plus 2.5 mM ATPγS. (B) Reactions with pBR322 χ° DNA (0.4 nM) contained ATP (5 mM) with or without NSAC1003 (200 μM). ✦, ✖ and Chi (χ⁺E): ATPγS-induced, NSAC1003-induced and Chi-dependent cleavage products, respectively.

Figure 6.. ATPγS-dependent cuts require RecD and the RecB nuclease active site.

(A) Purified RecBCD (0.3 or 0.1 nM) or RecBC (10 or 3.3 nM) was assayed with pBR322 χ⁺E224 DNA (0.4 nM) and either 5 mM ATP or 2.5 mM ATP plus 2.5 mM ATPγS and analyzed as in Fig. 2A. (B) As in panel A but with χ° DNA and purified RecB^D1080ACD (1 or 0.33 nM) instead of RecBC. ✦ and Chi (χ⁺E): ATPγS-induced and Chi-dependent cleavage products, respectively.