The RecB helicase-nuclease tether mediates Chi hotspot control of RecBCD enzyme

Abstract

In bacteria, repair of DNA double-strand breaks uses a highly conserved helicase-nuclease complex to unwind DNA from a broken end and cut it at specific DNA sequences called Chi. In Escherichia coli the RecBCD enzyme also loads the DNA strand-exchange protein RecA onto the newly formed end, resulting in a recombination hotspot at Chi. Chi hotspots regulate multiple RecBCD activities by altering RecBCD's conformation, which is proposed to include the swinging of the RecB nuclease domain on the 19-amino-acid tether connecting the helicase and nuclease domains. Here, we altered the tether and tested multiple RecBCD activities, genetically in cells and enzymatically in cell-free extracts. Randomizing the amino-acid sequence or lengthening it had little effect. However, shortening it by as little as two residues or making substitutions of ≥10 proline or ≥9 glycine residues dramatically lowered Chi-dependent activities. These results indicate that proper control of RecBCD by Chi requires that the tether be long enough and appropriately flexible. We discuss a model in which the swing-time of the nuclease domain determines the position of Chi-dependent and Chi-independent cuts and Chi hotspot activity.

Figure 1.

Models for RecBCD enzyme and its Chi-dependent promotion of DNA break repair and genetic recombination. (A) RecBCD pathway of genetic recombination (from (30)). RecBCD binds a ds DNA end (a) and unwinds the DNA with the production of enlarging ss DNA loops, because the RecD helicase moves faster than the RecB helicase (9) (b-c). If the 3′ and 5′ tails anneal, two loops are formed, but the tails may be kept apart by single-strand binding (SSB) protein (2,64). Upon encountering Chi, RecBCD cuts the strand with 5′ GCTGGTGG 3′ (5,6) (d), loads RecA onto the newly formed 3′ ss DNA end (8) (e) and later disassembles into the three inactive subunits (31) (f). The RecA-ssDNA filament invades intact ds DNA to form a D-loop (8) (f), which can be converted into a Holliday junction (g), which is resolved into reciprocal recombinants, or prime DNA replication (h), which produces a non-reciprocal recombinant and a parental-type DNA (1) (not shown). (B) Crystal structure of RecBCD bound to a ds DNA hairpin (12) (adapted from (30)). RecB (orange) contains helicase and nuclease domains connected by a 19-amino-acid tether (white star). RecD (green) is held to RecB via RecC (blue) and possibly by unstructured RecD amino acids not shown (35). The 3′-ended strand likely passes through a tunnel (yellow dashed line) in RecC and into the nuclease active site when Chi is encountered. (C) Signal-transduction model for Chi's control of RecBCD enzyme (from (30)). When Chi is in the RecC tunnel, RecC signals RecD to stop unwinding DNA. RecD then signals RecB to nick the DNA and to begin loading RecA. (D) Nuclease-swing model for Chi's control of RecBCD enzyme (adapted from (33)). Before DNA is bound, RecBCD in solution assumes its conformation in the published structures (12,35) (a). Upon binding DNA, the nuclease domain swings away from the exit of the RecC tunnel (33) (b). When Chi is encountered during unwinding, the nuclease domain swings back, cuts the DNA at Chi and begins loading RecA protein, perhaps after rotating to prevent further nuclease action (c).

Figure 2.

RecB (orange) with its tether connecting the helicase and nuclease domains. The ‘tether’ (amino acids 881–899 with 881–890 in salmon and 891–899 in red), defined here, is included in the ‘linker’ region (amino acids 870–940; gray), defined in refs. (12,35). The extents of three deletions studied here are shown. The linker also contains an α-helix (amino acids 913–922; yellow), defined in ref. (12). The RecC subunit (blue in ribbon representation) is in direct contact with much of RecB, including the tether.

Figure 3.

Chi hotspot activity in RecB tether mutants. Strains are transformants of strain V2831 (ΔrecBCD2731) with the indicated recB tether mutation on a plasmid (see Supplementary Table S2 for complete descriptions). Chi hotspot activity was determined as described (38) and is 5.1 ± 0.1 for recBCD⁺ (WT) and 0.99 ± 0.02 for recB21 (null mutant). Data are the mean of two experiments with the indicated range (|) or the mean ± SEM () from 3 to 17 independent experiments. Mutants are grouped by type: (A) deletion of amino acids; (B) addition of amino acids; (C) amino acid sequence changes; (D) substitutions with proline; (E) subsititutions with glycine.

Figure 4.

The RecB tether is required for full Chi-cutting activity but not enzyme assembly or DNA unwinding. Extracts were prepared from late log-phase cells of strain V2831 (ΔrecBCD2731) containing derivatives of plasmid pSA607 (recBC²⁷⁷³D) with the indicated recB tether deletion. (A) DNA unwinding and Chi cutting were assayed using HindIII-linearized ³²P-labeled pBR322 χ^o or χ⁺F225 DNA. The indicated amount of extract protein, per 15 μl reaction, was reacted with the DNA substrate (0.8 nM) at 37° for 2 min and the products separated on a 0.9% agarose gel. The positions of dsDNA substrate (DS), unwound ssDNA (SS; boiled), the product of Chi-dependent cutting (Chi), and limit-digest oligonucleotides (*) are shown. (B) RecB and RecC polypeptides and native forms of RecBCD enzyme in extracts of RecB tether mutants were separated on 3–8% Tris-acetate native or SDS denaturing polyacrylamide gels and detected by Western blots using the indicated polyclonal antibodies. Purified (p) RecBCD or RecBC enzyme was run as a marker. The lane with size markers (150, 120, and 65 kDa, top to bottom) is indicated (M). The migrations of RecB and RecC polypeptides and of RecBCD heterotrimer and RecBC heterodimer are indicated. Cross-reacting polypeptides present in extracts of V2831 containing pBR322 but lacking RecBCD are indicated (*). See Supplementary Figures S5 and S6 for additional data.

Figure 5.

Recombination proficiency in the RecBCD pathway is a linear function of Chi hotspot activity. Recombination proficiency was measured in Escherichia coli Hfr crosses (left) and lambda hotspot crosses (right). Data are relative to wt values and are colored according to the type of tether mutation in the key at top left. Data are from Tables 1 and 2, Supplementary Tables S4 and S6, and Figure 3. Regression lines are derived from least-squares analysis.