Strand Exchange Is Carried Out by the RecA/Rad51 Protein
Of all the steps of homologous recombination, strand exchange is the most difficult to imagine. How does the invading single strand rapidly sample a DNA duplex for homology? Once the homology is found, how does the exchange occur? How is the inherent stability of the template double helix overcome?
The answers to these questions came from biochemical and structural studies of the protein that carries out these feats, called RecA in E. coli and Rad51 in virtually all eukaryotic organisms. To catalyze strand exchange, RecA first binds cooperatively to the invading single strand, forming a protein–DNA filament that forces the DNA into an unusual configuration: groups of three consecutive nucleotides are held as though they were in a conventional DNA double helix but, between adjacent triplets, the DNA backbone is untwisted and stretched out (Figure 5–49). This unusual protein–DNA filament then binds to duplex DNA
Figure 5–48 Mechanism of double-strand break repair by homologous recombination. This is the preferred method for repairing DNA double-strand breaks that arise shortly after the DNA has been replicated, while the daughter DNA molecules are still held close together. In general, homologous recombination can be regarded as a flexible series of reactions, with the exact pathway differing from one case to the next. For example, the length of the repair “patch” can vary considerably depending on the extent of 5ʹ processing and new DNA synthesis, indicated in green.
in a way that stretches the duplex, destabilizing it and making it easy to pull the strands apart. The invading single strand then can sample the sequence of the duplex by conventional base-pairing. This sampling occurs in triplet nucleotide blocks: if a triplet match is found, the adjacent triplet is sampled, and so on. In this way, mismatches quickly lead to dissociation and only an extended stretch of base-pairing (at least 15 nucleotides) stabilizes the invading strand and leads to strand exchange.
RecA hydrolyzes ATP, and the steps described above require that each RecA monomer along the filament be in the ATP-bound state. However, the searching itself does not require ATP hydrolysis; instead, the process occurs by simple molecular collision, allowing many potential sequences to be rapidly sampled. Once the strand-exchange reaction is completed, however, ATP hydrolysis is necessary to disassemble RecA from the complex of DNA molecules. At this point, repair DNA polymerases and DNA ligase can complete the repair process, as shown in Figure 5–48.
Although accurately repairing double-strand breaks, which can arise from radiation or chemical reactions, is a crucial function of homologous recombination, perhaps its most important role is in rescuing stalled or broken DNA replication forks. Many types of events can cause a replication fork to break, and here we consider just one example: a single-strand nick or gap in the parental DNA helix just ahead of a replication fork. When the fork reaches this lesion, it falls apart—resulting in one broken and one intact daughter chromosome. The broken fork can be flawlessly repaired (Figure 5–50) using the same basic homologous recombination reactions we discussed above for the repair of double-strand breaks. With slight modifications, the set of reactions depicted in Figures 5–48 and 5–50— known collectively as homologous recombination—can accurately repair many different types of DNA damage.
Cells Carefully Regulate the Use of Homologous Recombination in DNA Repair
Although homologous recombination neatly solves the problem of accurately repairing double-strand breaks and other types of DNA damage, it does present