RecBCD - DNA Complex
David Marcey
© 2010

I. Introduction
II. RecBCD Subunit Structure
III. The RecBCD Complex

IV. References


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I. Introduction

The repair of double-strand breaks in DNA is essential for maintaining the integrity of the genetic material and for cell viability. The homologous recombination pathway serves as a primary means of double-strand break repair in eubacteria. An initial step in this pathway involves the RecBCD trimeric protein recognizing blunt-ended DNA at the site of a break, followed by complex processing of DNA inward from the break, finally resulting in a 3' overhang of single-stranded DNA (ssDNA) coated by RecA protein. This serves as the substrate for homologous recombination via the RecA pathway. The actions of RecBCD on a blunt-ended DNA molecule are outlined schematically in Figure 1.

Singleton, et al. (2004) obtained the crystal structure of the RecBCD heterotrimer of E. coli complexed with DNA, as shown at left. This structure yields insights into the multifaceted actions of RecBCD, and is the subject of this tutorial.

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II. RecBCD Subunit Structure

The RecB subunit is a 3'>>5' helicase, and its amino terminal region is composed of two domains, each having two subdomains (1A, 1B, 2A, 2B). These domains are similar in structure to other helicase superfamily 1 (SF1) members, although the RecB subdomain 1B contains an insertion of residues that form an extended arm that runs adjacent to, and contacts the duplex DNA (see below). The third, carboxy-terminal, nuclease domain, responsible for cleaving both the 3'-tailed strand and 5'-tailed strand (see Figure 1), is connected to the remainder of the RecB subunit by a long, 70 residue linker. Domains 1A and 2A display typical helicase motor domain structure, and drive the protein on ssDNA (up, in the orientation at left). The 3' tail of bound DNA is engaged by domain 2A in the crystal structure. The two tails of ssDNA would normally continue through the 3' and 5' tunnels of the RecBCD trimer, emerging at the nuclease domain of RecB, as discussed below.

The nuclease domain of the RecB subunit is similar in structure to the core of bacteriophage lambda exonuclease, and shares a conserved aspartate (asp1080) known to be involved in nuclease activity. Also found at the nuclease active site are two additional conserved acidic residues, glu1020 and asp1067, as well as a basic lys1082. In the heart of the active site is a Ca++ ion, which would normally be Mg++ in a catalytically active protein. This catalytically essential ion is coordinated by the two active site aspartates as well as by his956 and a main chain carbonyl of tyr1081. Although not thoroughly understood, the nuclease domain of RecB is also involved in the loading of RecA protein after processing of substrate DNA (see Figure 1) and prior to dissociation from it.

The RecC subunit, like RecB, contains three domains (1A, 1B, 2A, 2B, and 3). Domains 1 and 2 are structurally homologous to SF1 helicases, indicating that RecC may have evolved from a helicase ancestor. Further, Rigden (2005) discovered that domain 3 of RecC, connected to the remainder of the protein by a long linker, is structurally homologous to the RecB nuclease domain (and other nucleases like lambda exonuclease), although it is structurally incapable of nuclease activity. This domain has instead evolved the novel function of splaying the DNA duplex into single strands via residues on a prominent loop, the pin.

Several channels through the RecC protein serve important functions. These can best be observed with a surface view (allow ~20 seconds for surfaces to load):

(the next three animations require prior use of this button)

    • A large cavity serves to hold the 2B subdomain of RecB, providing a large surface area of subunit interaction. This cavity is bounded on one side by the linker that connects domains 3 and 2A.
    • The pin is observed to split the duplex. The 5' tail snakes through a tunnel in domain 3. This tail will be funneled to the RecD helicase on its way to the nuclease domain of RecB.
    • The RecC 3' channel, formed by the interface of subdomains 1A, 1B, 2A, 2B will accommodate the single-stranded 3' tail on its way to the nuclease domain of RecB.

The RecD subunit, composed of three domains (1, 2, and 3), is a 5'>>3' helicase of the SF1 superfamily. Domains 2 and 3 are similar to the structures of other SF1 helicases, although most of these are 3'>>5' helicases. Domain 1 provides substantial interaction with domain 2B of RecC.


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III. The RecBCD Complex

(the following animations in this section require prior use of this button - allow sufficient time to load surface view)

Extensive contacts are observed between the DNA and RecB and RecC atoms, rendered in spacefill. As mentioned previously, the RecC pin splits the DNA duplex into 5'- and 3'-tailed strands. The Arm of subdomain 1B of RecB contacts both strands of DNA ~12 base pairs downstream (up, in the view at left) from the pin-induced bifurcation. Upstream (down, in the view at left) from the split, The 3'- ending strand is fed to the RecB helicase and the 5'-ending strand is fed to the RecD helicase.

The ssDNA strands, produced by splitting of the duplex DNA at the RecC pin, thread through several channels on their way through the protein complex. The 5' channel, highlighted in a cutaway view by magenta flashing residues, accommodates the 5'-ending strand on its way to the nuclease domain of RecB, where strand cleavage takes place at the active site (marked by the Ca++ ion).

The 3'-ending strand passes along the 3' channel through the protein, exiting between subdomain 1A of RecC and the nuclease domain of RecB. A second exit would bring the 3'-ending strand past the active site of the nuclease, where digestion would occur. Mutations in RecC that perturb Chi sequence recognition (see Figure 1) alter residues at the opening of the active site exit channel.

Access to the nuclease active site is blocked by a loop in the crystal structure, comprising a short alpha helix flanked by long, flexible linkers. This flexible conformation would allow for regulation of strand access to the active site, thus potentially regulating nuclease activity. In the view at left, the RecC residues at the opening of the 3' active site exit channel are seen behind the loop, which lies above the active site.

Summary: The structural features discussed above provide insights into the complex activities of the RecBCD trimer as it processes blunt-ended DNA breaks (see Figure 1). The helicase motors of RecD and RecB pull the dsDNA into the pin of RecC, thus separating the duplex into 5'- and 3'-tailed single strands. The 3' tail follows a channel through the complex, emerging at the nuclease active site. The 5' tail is threaded through a different channel, also emerging near the nuclease active site. The initial, more frequent cleavage of the 3'-tailed strand by RecBCD, compared to the 5'-tailed strand, may be attributed to the 3' strand being better positioned for access to the nuclease active site. After encountering a Chi site, cleavage of the 3' strand ceases, perhaps because the 3' active site exit channel is blocked by a flexible loop. This then would allow for an increased frequency of cleavage of the 5' strand. Loading of RecA on the 3' strand by RecBCD then occurs.


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IV. References

Rigden, D.J.: An inactivated nuclease-like domain in RecC with novel function: implications for evolution. BMC Structural Biology 5:9 (2004).

Singleton, M.R., Dillingham, M.S., Gaudler, M., Kowalczykowski, S.C., Wigley, D.B.: Crystal Structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 432: 187-193 (2004).

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Acknowledgement: The format of this web page is modified from a template provided by Dr. Angel Herraez, Bioquimica y Biologia Molecular, Universidad de Alcala, E-28871, Alcala de Henares (Madrid), Spain.