Homologous recombination in DNA repair and DNA damage tolerance

Abstract

Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks (DSBs) and interstrand crosslinks (ICLs). In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR: homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.

Figure 1

Pathways of recombination in DSB repair. Homologous recombination can be conceptually divided into three stages: presynapsis, synapsis, and postsynapsis. During presynapsis, DSB ends are recognized and processed to a 3′-OH ending single-stranded tail (steps 1–2). In synapsis, DNA strand invasion by the Rad51-ssDNA filament generates a D-loop (step 3). At least three different pathways are proposed after the D-loop intermediate. In synthesis-dependent strand annealing (SDSA, steps 4a – 5a – 6a), the invading strand is disengaged after DNA synthesis and annealed with the second end, leading to localized conversion without crossover. This process may involve multiple rounds of invasion, synthesis, and disengagement. In break-induced replication (BIR, steps 4a – 5b – 6b), the D-loop is assembled into a full replication fork, copying the entire distal part of the chromosome to result in loss-of heterozygosity (LOH). In double-strand break repair (DSBR, steps 4b – 5c – 6c–e – 7), both ends of the DSB are engaged, either by independent strand invasion or by second end capture, leading to double Holliday junction formation. The junction can be processed by either a resolvase into non-crossover or crossover products (steps 6c and d) or dissolved by a mechanism involving BLM-mediated branch migration and TOPOIIIα-catalyzed dissolution of a hemicatenane (step 6e), leading exclusively to non-crossover products (step 7).

Figure 2

Pathways of homologous recombination at stalled/broken replication forks. (A) Possible pathways resolving leading-strand blockage. Stalled replication forks (step 1) can be cleaved by an endonuclease to generate a one-sided DSB. One-sided DSB repair by recombination (steps 2a – 3a – 4a – 5a) proceeds in analogy to BIR (Figure 1) involving DNA strand invasion and re-establishment of a functional fork. A single Holliday junction is created in the process. The initial blocking lesion must be either repaired or bypassed by TLS polymerases. TLS can also lead to direct bypass of the original blocking lesion (step 3b, green line). Uncoupling of lagging-strand synthesis (step 2b) can lead to direct lesion bypass by TLS (step 3b) or downstream re-initiation of leading strand synthesis, resulting in a leading strand gap, which can be repaired by recombination (steps 3c – 4c – 5c). The different strand invasion pathways are not detailed here, but are shown below for lagging strand gaps (see b; steps 2a – 3a – 4 and 2b – 3b – 4). The lesion may be repaired later or is tolerated. Alternatively, the fork could regress into a Holliday junction (also called a chicken foot), where the nascent lagging strand serves as a template for the leading strand (steps 3d – 4d). Reversal of the chicken foot enables fork progression, and the blocking lesion may be repaired later or is tolerated (step 5d). (B) Possible pathways resolving lagging strand blockage. Downstream re-initiation of lagging-strand synthesis after blockage leaves a gap on the lagging strand (step 1), which can be repaired by recombination (steps 2a – 3a/b – 4). Initiation from the uninterrupted strand of the gap (step 2a) leads to formation of a paranemic joint that involves partial Holliday junctions and possible double Holliday junctions. Initiation by the 3′ end strand of the interrupted strand generates a D-loop (2b – 3b). The blockage can also be directly bypassed by TLS (step 2c; green line).

Figure 3

Homologous recombination and repair of DNA interstrand crosslinks. Possible pathways to resolve replication forks stalled at interstrand crosslinks. The stalled replication fork is recognized and cleaved by a specific endonuclease (hMus81-Eme1 [144]) in the leading-strand template to generate a one-sided DSB (steps 1–3). Introduction of a second incision on the other side of the ICL (step 4a) allows the lesion to flip out and to be bypassed by TLS (green line). The DSB is processed to form a 3′-OH ending single-stranded tail (step 5a) and to initiate DNA strand invasion (step 6a). The replication fork is restored (steps 7a) and the lesion is bypassed by TLS (green line). The lesion is eventually repaired, either after HR as drawn in step 8 or before (e.g. at step 5a). The DSB can also initiate DNA strand invasion using the homolog as a template (step 4b). DNA is synthesized across the lesion region (step 5b), disengaged (step 6b) and reinvasion of the sister chromatid behind the lesion site can lead to restoration of the replication fork and tolerance of the lesion (7b; the step from D-loop to recovered fork are not drawn and equivalent to Figure 2A, steps 3a–4a–5a). The hypothetical steps for ICL repair by nucleotide excision repair are not drawn here. For additional schemes for ICL repair/tolerance at stalled replication forks or in non-replicating DNA see refs. [138, 139, 145].