How RecBCD enzyme and Chi promote DNA break repair and recombination: a molecular biologist's view

Abstract

The repair of DNA double-strand breaks (DSBs) is essential for cell viability and important for homologous genetic recombination. In enteric bacteria such as Escherichia coli, the major pathway of DSB repair requires the RecBCD enzyme, a complex helicase-nuclease regulated by a simple unique DNA sequence called Chi. How Chi regulates RecBCD has been extensively studied by both genetics and biochemistry, and two contrasting mechanisms to generate a recombinogenic single-stranded DNA tail have been proposed: the nicking of one DNA strand at Chi versus the switching of degradation from one strand to the other at Chi. Which of these reactions occurs in cells has remained unproven because of the inability to detect intracellular DNA intermediates in bacterial recombination and DNA break repair. Here, I discuss evidence from a combination of genetics and biochemistry indicating that nicking at Chi is the intracellular (in vivo) reaction. This example illustrates the need for both types of analysis (i.e., molecular biology) to uncover the mechanism and control of complex processes in living cells.

Fig 1

Growth cycle of phage λ. Duplex DNA is represented as a single line. Linear DNA (top left) in the viral particles is injected into cells, where the cohesive ends (cos) are ligated to form circular DNA. Early replication in the theta (θ) mode produces monomeric circles, which must be converted to concatemeric DNA to be packaged (matured) into viable phage particles. This can occur by late rolling-circle (σ) replication, but only when the RecBCD nuclease is absent, because of mutation or inhibition by the λ Gam protein. Alternatively, recombination by the λ Red pathway or the E. coli RecBCD pathway can convert monomeric circles into dimeric or higher-order concatemers containing two or more cos sites required for packaging. RecBCD gains access to a monomeric circle when cos is cut during maturation; packaging proteins bound at the left end block access, forcing RecBCD to enter the right end and travel leftward. Chi stimulates RecBCD-promoted recombination, thereby allowing more λ red gam mutant DNA to be packaged, with the formation of larger plaques than those formed without Chi. (Modified from reference [copyright 1983, Cold Spring Harbor Laboratory Press].)

Fig 2

Conventional genetic map of λ. Mutations creating Chi were mapped to four sites (χ⁺A to χ⁺D) and in plasmid pBR322 inserted into the site indicated between χ⁺B and χ⁺C. Each of these mutations contains the Chi sequence, 5′-GCTGGTGG-3′, on the top strand, as indicated. (The sequence of χ⁺A has not to my knowledge been reported.) The scheme is drawn to scale on the 48.5-kb λ DNA.

Fig 3

DNA-unwinding intermediates made by RecBCD. Electron micrographs of DNA after a brief reaction with RecBCD enzyme reveal loop-tails (a) and twin loops (“rabbit ears”) (b). ssDNA, bound by SSB, is thick, and dsDNA is thin in these preparations. The single-strand tails of loop-tails can anneal to form twin loops (Fig. 4 and 7) (see reference 110).

Fig 4

Early model for recombination based on nicking at Chi. Solid lines, DNA of one parent; dashed lines, DNA of the other parent. (A to D) RecBCD, then called RecBC, unwinds DNA (A to C) as shown in Fig. 3 and nicks one strand at Chi (D). (E to G) The newly generated end is elongated by continued unwinding (E) and is bound by RecA protein (F), which forms a D-loop with an intact, homologous duplex (G). (H to J) Cutting of the displaced strand in the D-loop allows that strand to pair with the gap in the Chi-containing parent to form a Holliday junction (H), which is resolved into either a noncrossover (I) or a crossover (J). (Reproduced from reference with permission of the publisher.)

Fig 5

Alternative reactions of purified RecBCD during unwinding of DNA with Chi. With excess ATP (left), RecBCD nicks the top strand containing Chi (5′-GCTGGTGG-3′). With excess Mg²⁺ ions (right), RecBCD endonucleolytically cleaves the top (3′-ended) strand up to Chi, cuts the bottom strand, and endonucleolytically cleaves that strand beyond Chi.

Fig 6

Model for joint molecule formation based on degradation up to Chi (Fig. 5, right). (A and B) RecBCD unwinds DNA, as shown in Fig. 3; SSB binds the single-strand loop and tail. (C) At Chi, degradation switches from the top to the bottom strand, and RecBCD begins to load RecA onto the 3′-ended strand with Chi near its end. (D to F) This RecA-ssDNA filament (D and E) invades intact DNA to form a D-loop (F). (Reprinted from reference with permission of Elsevier.)

Fig 7

Model for recombination based on nicking at Chi (Fig. 5, left). This is an expanded version of the model shown in Fig. 4, with thick lines representing one parent. The D-loop (f) can be converted into a Holliday junction (g) and resolved into a crossover (shown) or a noncrossover (not shown here). Alternatively, the 3′ end of the invading Chi tail can prime DNA replication (h); the cutting of strands (open arrowheads), swapping of strands, and ligation produce one crossover-type recombinant but not its reciprocal, plus one parental-type molecule (not shown). This mechanism is also called “break-induced replication” (BIR). (Reprinted from reference with permission [copyright 2007, Cold Spring Harbor Laboratory Press].)

Fig 8

Proposal for formation of high-molecular-weight (HMW) DNA during rolling-circle (σ) replication. RecBCD initiates unwinding at the open end of the rolling circle (Fig. 1). At a properly oriented Chi site, it promotes the recombination of the replicating DNA with an intact circle to form a dumbbell-shaped molecule. Divergent replication from each fork elongates the DNA connecting the circles, which migrates slowly during gel electrophoresis and is measured as HMW DNA (see reference 31).

Fig 9

Recombination of Chi⁺ DNA versus degradation of Chi^o DNA by RecBCD. Linear DNA is unwound by RecBCD (Fig. 3). If the DNA contains a Chi site, RecBCD nicks the DNA and loads RecA protein onto the newly generated 3′ end, which then engages intact DNA and undergoes recombinational repair (Fig. 7). If the DNA does not contain Chi, the ssDNA is degraded to short oligonucleotides by RecBCD's single-strand (ss) exonuclease activity. If the DNA cannot recombine, as in a recA mutant or in the absence of sufficient nucleotide sequence identity, Chi⁺ DNA is also degraded.

Fig 10

Crystal structure of RecBCD bound to hairpin-shaped DNA (PDB accession number 1W36 [89]). RecB is orange, RecC is blue, and RecD is green. Four base pairs of the DNA (multicolored) are unwound, with the 3′ end in the RecB helicase domain and the 5′ end in RecC headed toward the helicase domain of RecD. During unwinding, the 3′ end may pass through a tunnel (dashed yellow line) in RecC, where Chi may be recognized, and be cut by the RecB nuclease domain.

Fig 11

Model for the regulation of RecBCD by Chi. When Chi on the 3′-ended strand passes through the tunnel in RecC (Fig. 10), RecC signals RecD, the faster helicase, to stop. When stopped, RecD signals RecB to cut the DNA at Chi and begin loading RecA protein onto the newly generated 3′ end with Chi (Fig. 7). (Reprinted from reference with permission [copyright 2007, Cold Spring Harbor Laboratory Press].)