Cells are under constant genotoxic pressure from both endogenous and exogenous sources. It has been estimated that more than tens of thousands of DNA lesions occur in a single human cell every day (1
). These lesions need to be repaired to avoid deleterious mutations, blockage of replication and transcription, and chromosomal breakage. The importance of DNA repair to human health is highlighted by the fact that failure to repair damaged DNA increases the likelihood of developing tumours and other diseases. In this review, we focus on homologous recombination (HR), a mechanism that repairs a variety of DNA lesions, including double-strand DNA breaks (DSBs), single-strand DNA gaps and interstrand crosslinks. Among these lesions, DSBs are highly toxic as a single unrepaired DSB can lead to aneuploidy, genetic aberrations or cell death. DSBs can be generated by a number of sources, including treatment with genotoxic chemicals and ionizing radiation, collapsed replication forks, and other endogenous DNA breaks. On the other hand, repair of DSBs is essential for the first meiotic division where it contributes to the formation of chiasmata, required for proper pairing and segregation of homologous chromosomes, and the generation of genetic diversity in most organisms (2
A central player in HR is the strand-exchange protein, called Rad51 in eukaryotic cells (RecA in Escherichia coli
). Rad51 functions in all three phases of HR: presynapsis, synapsis and post-synapsis [A, (3
)]. In the presynaptic phase, Rad51 is loaded onto single-strand DNA (ssDNA) that either is generated by degrading 5′-strands at DSBs or arises from replication perturbation. The resulting Rad51–ssDNA filament (presynaptic filament) is right-handed and comprises six Rad51 molecules and 18 nucleotides per helical turn. The ssDNA within the filament is stretched as much as half the length of B-form dsDNA (4
). The stretching of the filament is essential for fast and efficient homology search (5
). During synapsis, Rad51 facilitates the formation of a physical connection between the invading DNA substrate and homologous duplex DNA template, leading to the generation of heteroduplex DNA (D-loop). Here, Rad51–dsDNA filaments are formed by accommodating both the invading and donor ssDNA strands within the filament. Finally, during post-synapsis when DNA is synthesized using the invading 3′-end as a primer, Rad51 dissociates from dsDNA to expose the 3′-OH required for DNA synthesis.
Figure 1. Models for the repair of DNA double-strand breaks. DNA DSBs are resected to generate 3′-protruding ends followed by formation of Rad51 filaments that invade into homologous template to form D-loop structures. (A) After priming DNA synthesis, three (more ...)
At least three different routes can be used once DNA synthesis is initiated (B–D). First, as envisioned in the double-strand break repair model (DSBR), the second end of DSB can be engaged to stabilize the D-loop structure (second-end capture), leading to the generation of a double-Holliday Junction (dHJ) [(7
), reviewed in Ref. (8
); B]. A dHJ is then resolved to produce crossover or non-crossover products (B) or dissolved to exclusively generate non-crossover products. Second, the invading strand can be displaced from the D-loop and anneals either with its complementary strand as in gap repair or with the complementary strand associating with the other end of the DSB. This represents the synthesis-dependent strand-annealing mode of HR (SDSA) [(9
), C]. SDSA mechanism is preferred over DSBR during mitosis. During meiosis, crossovers are formed by resolution of dHJs via the DSBR mechanism, while non-crossovers are primarily produced via SDSA mechanism (10
). In the third mode, the D-loop structure can assemble into a replication fork and copy the entire chromosome arm in a process called break-induced replication (BIR) [(12
), D]. This mechanism is evoked more often when there is only one DNA end, either due to the loss of the other end or in the process of lengthening telomeres in telomerase-deficient cells.
All the above pathways require Rad51, with the exception of some forms of BIR. However, DSBs can also be sealed by pathways independent of Rad51 (E and F). One of these pathways is the single-strand annealing pathway (SSA). In SSA, ssDNA sequences generated during DSB processing contain regions of homology at both sides of DSB and can be annealed and ligated [(13
), E]. SSA does not require Rad51 but requires other HR proteins that mediate annealing. Another Rad51-independent pathway that operates at DSBs is non-homologous end joining (NHEJ), which ligates ends of DSBs with little or no requirement for homology [reviewed in Ref. (8