Figure 5–49 Strand invasion catalyzed by the RecA protein. Our understanding of this reaction is based in part on structures determined by x-ray diffraction studies of RecA bound to singleand double-strand DNA. These DNA structures (shown without the RecA protein) are on the left side of the diagram. Starting at the top, ATP-bound RecA associates with single-strand DNA, holding it in an elongated form where groups of three bases are separated from each other by a stretched and twisted backbone. In the next step, the RecA-bound single strand then binds to duplex DNA, destabilizing it and allowing the single strand to sample its sequence through base-pairing, three bases at a time. If no match is found, the RecA-bound single strand of DNA rapidly dissociates and begins a new search. If an extensive match is found, the structure is disassembled through ATP hydrolysis, resulting in the dissociation of RecA and the exchange of one single strand of DNA for another, thereby forming a heteroduplex. (PDB code: 3CMX.)
Figure 5–50 Repair of a broken replication fork by homologous recombination. When a moving replication fork encounters a single-strand break, it will collapse, but can be repaired by homologous recombination. The process uses many of the same reactions shown in Figure 5–48 and proceeds through the same basic steps. Green strands represent the new DNA synthesis that takes place after the replication fork has broken. This pathway allows the fork to move past the site that was nicked on the original template by using the undamaged duplex as a template to synthesize DNA. (Adapted from M.M. Cox, Proc. Natl Acad. Sci. USA 98:8173–8180, 2001. With permission from National Academy of Sciences.) some dangers to the cell as it sometimes “repairs” damage using the wrong bit of the genome as the template. For example, sometimes a broken human chromosome is “repaired” using the homolog from the other parent instead of the sister chromatid as the template. Because maternal and paternal chromosomes differ in DNA sequence at many positions along their lengths, this type of repair can convert the sequence of the repaired DNA from the maternal to the paternal sequence or vice versa. The result of this type of errant recombination is known as loss of heterozygosity. It can have severe consequences if the homolog used for repair contains a deleterious mutation, because the recombination event destroys the “good” copy. Loss of heterozygosity, although rare, is a critical step in the formation of many cancers (discussed in Chapter 20).
Cells go to great lengths to minimize the risk of mishaps of these types; indeed, nearly every step of homologous recombination is carefully regulated. For example, the first step, processing of the broken ends, is coordinated with the cell cycle: the nuclease enzymes that carry out this process are activated (in part, by phosphorylation) only in the S and G2 phases of the cell cycle, when a daughter duplex (either as a partially replicated chromosome or a fully replicated sister chromatid) can serve as a template for repair (see Figure 5–50). The close proximity of the two daughter chromosomes disfavors the use of other genome sequences in the repair process.
The loading of RecA or Rad52 onto the processed DNA ends and the subsequent strand-exchange reaction are also tightly controlled. Although these proteins alone can carry out these steps in vitro, a series of accessory proteins, including Rad52, is needed in eukaryotic cells to ensure that homologous recombination is efficient and accurate (Figure 5–51). There are many such accessory proteins, and exactly how they coordinate and control homologous recombination remains a mystery. We do know that the enzymes that catalyze recombinational repair are made at relatively high levels in eukaryotes and are dispersed throughout the nucleus in an inactive form. In response to DNA damage, they rapidly converge on the sites of DNA damage, become activated, and form “repair factories” where many lesions are apparently brought together and repaired (Figure 5–52).