DNA Recombination:- Recombination Requires a Host of Enzymes and Other Proteins
Enzymes that promote various steps of homologous re combination have been isolated from both prokaryotes and eukaryotes. In E. coli, the recB, recC, and recD genes encode the RecBCD enzyme, which has both helicase and nuclease activities. The RecA protein promotes all the central steps in the homologous recombination process: the pairing of two DNAs, formation of Holliday intermediates, and branch migration (as described below). The RuvA and RuvB proteins (repair of UV damage) form a complex that binds to Holliday in termediates, displaces RecA protein, and promotes branch migration at higher rates than does RecA. Nucleases that specifically cleave Holliday intermediates, often called resolvases, have been isolated from bacteria and yeast. The RuvC protein is one of at least two such nucleases in E. coli.
The RecBCD enzyme binds to linear DNA at a free (broken) end and moves inward along the double helix, unwinding and degrading the DNA in a reaction coupled to ATP hydrolysis (Fig. 1). The activity of the en zyme is altered when it interacts with a sequence re ferred to as chi, (5)GCTGGTGG. From that point, degradation of the strand with a 3 terminus is greatly reduced, but degradation of the 5-terminal strand is increased. This process creates a single-stranded DNA with a 3 end, which is used during subsequent steps in recombination . The 1,009 chi sequences scattered throughout the E. coli genome enhance the frequency of recombination about five- to tenfold within 1,000 bp of the chi site. The enhancement declines as the distance from the site increases. Sequences that en hance recombination frequency have also been identified in several other organisms. RecA is unusual among the proteins of DNA metabolism in that its active form is an ordered, helical fil ament of up to several thousand RecA monomers that assemble cooperatively on DNA (Fig. 2). This filament normally forms on single-stranded DNA, such as that produced by the RecBCD enzyme. The filament will also form on a duplex DNA with a single-strand gap; in this case, the first RecA monomers bind to the single stranded DNA in the gap, after which the assembled filament rapidly envelops the neighboring duplex. The RecF, RecO, and RecR proteins regulate the assembly and disassembly of RecA filaments.
A useful model to illustrate the recombination ac tivities of the RecA filament is the in vitro DNA strand exchange reaction (Fig. 3). A single strand of DNA is first bound by RecA to establish the nucleoprotein fil ament. The RecA filament then takes up a homologous duplex DNA and aligns it with the bound single strand. Strands are then exchanged between the two DNAs to create hybrid DNA. The exchange occurs at a rate of 6 bp/s and progresses in the 5→3 direction relative to the single-stranded DNA within the RecA filament. This reaction can involve either three or four strands (Fig. 3); in the latter case, a Holliday intermediate forms during the process.
As the duplex DNA is incorporated within the RecA filament and aligned with the bound single-stranded DNA over regions of hundreds of base pairs, one strand of the duplex switches pairing partners (Fig. 4, step 2). Because DNA is a helical structure, continued strand exchange requires an ordered rotation of the two aligned DNAs. This brings about a spooling action (steps 3 and 4) that shifts the branch point along the helix. ATP is hydrolyzed by RecA protein during this reaction. Once a Holliday intermediate has formed, a host of enzymes—topoisomerases, the RuvAB branch migration protein, a resolvase, other nucleases, DNA polymerase I or III, and DNA ligase—are required to complete re combination. The RuvC protein (Mr 20,000) of E. coli cleaves Holliday intermediates to generate full-length, unbranched chromosome products.
FIGURE 1 Helicase and nuclease activities of the RecBCD en zyme. Entering at a double-stranded end, RecBCD unwinds and de grades the DNA until it encounters a chi sequence. The interaction with chi alters the activity of RecBCD so that it generates a single stranded DNA with a 3 end, suitable for subsequent steps in recombination. Movement of the enzyme requires ATP hydrolysis. This en zyme is believed to help initiate homologous genetic recombination in E. coli. It is also involved in the repair of double-strand breaks at collapsed replication forks.
FIGURE 2 RecA. (a) Nucleoprotein filament of RecA protein on single-stranded DNA, as seen with the electron microscope. The striations indicate the right-handed helical structure of the filament. (b) Surface contour model of a 24-subunit RecA filament. The filament has six subunits per turn. One subunit is colored red to provide perspective (derived from PDB ID 2REB).
FIGURE 3 DNA strand-exchange reactions promoted by RecA protein in vitro. Strand exchange involves the separation of one strand of a duplex DNA from its complement and transfer of the strand to an alternative complementary strand to form a new duplex (heteroduplex) DNA. The transfer forms a branched intermediate. Formation of the final product depends on branch migration, which is facilitated by RecA. The reaction can involve three strands (left) or a reciprocal exchange between two homologous duplexes—four strands in all (right). When four strands are involved, the branched intermediate that results is a Holliday intermediate. RecA protein promotes the branch-migration phases of these reactions, using energy derived from ATP hydrolysis.
FIGURE 4 Model for DNA strand exchange mediated by RecA protein. A three-strand reaction is shown. The balls representing RecA protein are undersized relative to the thickness of DNA to clarify the fate of the DNA strands. 1 RecA protein forms a filament on the single-stranded DNA. 2 A homologous duplex incorporates into this complex. 3 As spooling shifts the three-stranded region from left to right, one of the strands in the duplex is transferred to the single strand originally bound in the filament. The other strand of the duplex is dis placed, and a new duplex forms within the filament. As rotation con tinues ( 4 and 5), the displaced strand separates entirely. In this model, hydrolysis of ATP by RecA protein rotates the two DNA molecules relative to each other and thus directs the strand exchange from left to right as shown.
