Chi hotspot activity in Escherichia coli without RecBCD exonuclease activity: implications for the mechanism of recombination

Abstract

The major pathway of genetic recombination and DNA break repair in Escherichia coli requires RecBCD enzyme, a complex nuclease and DNA helicase regulated by Chi sites (5'-GCTGGTGG-3'). During its unwinding of DNA containing Chi, purified RecBCD enzyme has two alternative nucleolytic reactions, depending on the reaction conditions: simple nicking of the Chi-containing strand at Chi or switching of nucleolytic degradation from the Chi-containing strand to its complement at Chi. We describe a set of recC mutants with a novel intracellular phenotype: retention of Chi hotspot activity in genetic crosses but loss of detectable nucleolytic degradation as judged by the growth of mutant T4 and lambda phages and by assay of cell-free extracts. We conclude that RecBCD enzyme's nucleolytic degradation of DNA is not necessary for intracellular Chi hotspot activity and that nicking of DNA by RecBCD enzyme at Chi is sufficient. We discuss the bearing of these results on current models of RecBCD pathway recombination.

Figure 1.—

Two reactions of purified RecBCD enzyme at Chi, depending on the ATP:Mg²⁺ ratio. (Top) RecBCD binds to a dsDNA end, the RecB helicase being engaged with the 3′-end and the RecD helicase with the 5′-end. RecD is faster than RecB, and a ssDNA loop accumulates on the 3′ → 5′ (“top”) strand ahead of the RecB subunit (A). (Left) If the concentration of ATP is greater than that of Mg²⁺, RecBCD nuclease activity is low, and the enzyme nicks the top strand at Chi (B). (Right) If the concentration of Mg²⁺ is greater than that of ATP, RecBCD nuclease activity is high, and the enzyme degrades the top strand up to Chi (B), nicks the bottom strand, and degrades the bottom strand to the left of Chi (C). RecBCD loads RecA protein onto the top strand to the left of Chi (the “Chi tail”) (C). See Introduction and discussion for further description and references.

Figure 2.—

Structure of RecBCD bound to a dsDNA end (after Singleton et al. 2004). The RecB subunit is orange, RecC is blue, and RecD is green. (A) Surface representation. The 4 terminal base pairs of DNA are unwound, and the 3′-end lies within RecB. During unwinding, this strand is postulated to pass through a tunnel in RecB and RecC on its way to the nuclease domain of RecB. Chi is postulated to be recognized by parts of the tunnel in RecC (red arrow). The 5′-end of the DNA lies within RecC and extends toward an unordered part of RecD lying behind the surface shown. (B) Ribbon representation. The C terminus of RecC is red (residues 790–840) or magenta (residues 841–1122) to indicate the region deleted by recC2717 or truncated by the recC1041 nonsense mutation (see Figure 3). These mutants have the Chi⁺ Nuc⁻ Rec⁺ (†) phenotype (see results). Trp⁸⁴¹ lies behind a magenta helix but in front of the DNA (light blue line). The C-terminal domain of RecC may contact the unordered part of RecD (ends marked with black dots) and aid assembly of RecD into the holoenzyme complex (Amundsen et al. 2002). The orange dot near the bottom indicates the Ca²⁺ ion crystallized in the nuclease active site. Relative to the view in A, the top of the molecule is tilted slightly forward and rotated slightly clockwise, as viewed from the top. Images were constructed with PyMol and data for RecBCD from the Protein Databank (accession code 1W36).

Figure 3.—

Intermediate length C-terminal deletions of recC retain Chi hotspot activity. Open bars indicate the extent of the RecC polypeptide remaining in each exonuclease III-generated deletion. The recC1041 nonsense mutation is included for reference. Chi activity and λ recombinant frequencies are the mean ± SEM from five independent experiments with plasmid-bearing derivatives of strain V68 (recC73) or strain V2830 (ΔrecC2730∷kan). The recombinant frequencies in Hfr crosses are from two independent experiments with plasmid-bearing derivatives of strain V68. The bar at the bottom indicates the positions of the recC73 frameshift mutation, the recC1010 missense mutation, and the left end of the Δ(recC–argA)234 deletion.

Figure 4.—

Enzyme from recC1041 cells has DNA-unwinding and Chi-cutting activities. RecBCD, RecBC, and RecBC¹⁰⁴¹ enzyme were assayed using HindIII-linearized, 5′ ³²P-labeled pBR322 χ⁺F225 or χ⁰ DNA (Amundsen et al. 2000). The DNA substrate (0.8 nm) and indicated concentration of purified enzyme were incubated at 37° for 3 min. The reaction products were analyzed by electrophoresis in a 1% agarose gel. The positions of dsDNA substrate (DS), unwound ssDNA (SS; boiled), and the major product of Chi-dependent nicking (Chi) are shown.

Figure 5.—

Nick-at-Chi model of RecBCD-promoted recombination (after Smith et al. 1981b; Smith 2001). RecBCD binds a dsDNA end (A) and unwinds DNA to produce a loop-tail structure (B) (Figures 1 and 2). The ssDNA ends can anneal to form a “rabbit ear” structure (C). At Chi (small shaded circles), RecBCD nicks the top strand (D) and loads RecA onto the 3′ Chi tail (E) (Figure 1), which undergoes strand exchange with a homologous duplex to form a D-loop (F). At some point after Chi, RecBCD leaves the DNA and the three subunits disassemble. Two fates of the D-loop joint molecule are shown at the bottom. (Bottom left) Cutting of the D-loop and reciprocal strand exchange form a Holliday junction (G), whose migration and resolution (involving cutting at Δ, strand swapping, and ligation) by some combination of RuvABC and RecG proteins produce reciprocal recombinants. (Bottom right) The 3′ Chi tail primes leading-strand DNA synthesis, which is converted into a replication fork (H); resolution at Δ produces one recombinant, plus one parental and one fragmented molecule not shown. This break-copy scheme is identical to break-induced replication (BIR). Alternative resolutions are not shown.