DNA Repair: The Search for Homology

Abstract

The repair of chromosomal double-strand breaks (DSBs) by homologous recombination is essential to maintain genome integrity. The key step in DSB repair is the RecA/Rad51-mediated process to match sequences at the broken end to homologous donor sequences that can be used as a template to repair the lesion. Here, in reviewing research about DSB repair, I consider the many factors that appear to play important roles in the successful search for homology by several homologous recombination mechanisms. See also the video abstract here: https://youtu.be/vm7-X5uIzS8.

Keywords: DNA strand invasion; Rad51/RecA; double-strand break repair; homologous recombination; search for homology.

Figure 1.

Single Strand Annealing (SSA). A. A DSB created between flanking repeated sequences (blue boxes) in direct orientation is resected by 5’ to 3’ exonucleases until complementary sequences are exposed, so that they can anneal with the aid of proteins such as Rad52. Nonhomologous 3’ tails are clipped off by the Rad1^XPF-Rad10^Ercc10 endonuclease complex and gaps are filled in to create a deletion. In the process, single stranded sequences that might provoke strand invasion and recombination elsewhere in the genome (light orange box) and interfere with the completion of SSA. B. Competition between alternative SSA events [1]. After induction of pairs of DSBs separated by about 1 kb, the ends are resected, allowing two viable alternative outcomes: either a pair of deletions or a pair of reciprocal translocations. Apparently because the sequences needed for the reciprocal translocation are rendered single-stranded more rapidly, translocations predominate over deletions.

Figure 2.

Modes of homologous recombination (HR). A. DSB repair by synthesis-dependent strand annealing (SDSA) creates a small patch of newly copied DNA to repair the DSB without an accompanying crossover. Here, strand invasion leads to the copying of the template, but the new strand is displaced from the donor and is captured by the second end of the DSB. This leads to another round of DNA synthesis and repair of the break. B. DSB repair through a double-Holliday junction (dHJ) intermediate engages both ends of the DSB with the donor sequences, creating a pair of Holliday junctions. After new DNA synthesis to fill in the gaps, the dHJ can either be “dissolved” by Sgs1^BLM-Rmi1-Top3^Top3α to produce an outcome similar to that in (A) or can be cleaved by structure-specific nucleases to create a crossover accompanying DSB repair. C. Break-induced replication (BIR) occurs when only one end of the DSB shares sufficient homology with other sequences in the genome. Repair occurs through a migrating “D-loop” in which the second strand of new synthesis is added only after some delay.

Figure 3.

Ectopic repair of a DSB is influenced by proximity and possibly by DNA damage-stimulated changes in chromatin structure and chromosome movement. A. A cartoon of a budding yeast nucleus, with chromosomes tethered by their centromeres at the spindle pole body and displaying a so-called Rabl orientation. A DSB (red) causes both local (yellow circles) and a weaker global (light green circle) changes in chromatin structure and chromosome mobility. Image based on data and illustrations by Zimmer and Fabre [104]. B. The efficiency of intrachromosomal and interchromosomal repair, using donors of a few kb, is strongly influenced by the proximity - as measured by contact frequency [31] - when the efficiency of DSB repair, measured by viability, was compared for 20 budding yeast strains, each with a donor in a different interchromosomal location [33].

Figure 4.

A. A Cis/Trans test of how DSB ends are linked together. Experiments by Jain et al. [2]. I. A DSB induced between two halves of a LEU2 gene (LE and U2) can be repaired by gene conversion with an unbroken LEU2 sequence on a different chromosome. Here, the two ends participating in repair are in Cis. A second DSB is repaired by single-strand annealing between flanking URA3 sequences. II. In Trans, the same two types of repair must occur as in (A), but now the LE and U2 ends are derived from two DSBs. The opposite DSB ends are repaired by SSA between URA3 sequences. III. Repair of LEU2 in trans is markedly slower than in Cis, but this delay can be suppressed by deleting one of the URA3 segments, so that there is no competition between the two ends of one DSB to participate in two different repair events (IV). B. Sequences close to the end of a DSB are not used preferentially. A DSB can either be repaired by gene conversion with an ectopic URA3 or LYS2 donor [92]. The DSB ends were perfectly matched, except for a single base pair substitution, to a mutated HO cleavage site (black vertical bar). Both outcomes are frequent.

Figure 5.

Template switching between divergent, unlinked donors during gap repair initiated by a DSB. Based on the observations of Hicks et al. [105] and Tasponina and Haber [102]. The sequence of events is that expected for gene conversion by SDSA. Black sequences are homologous and repair involves copying donor sequences. Left: simple SDSA results in the copying of the blue sequences from a single donor. Right: Template switching creates a blue/green/blue chimeric sequence. Note that the assay requires that there be two jumps to allow the completion of the DSB repair event.