Figure 1. Homologous recombination pathway
Overview of homologous recombination pathway
Cells are under constant genotoxic pressure from both endogenous and exogenous sources, which may cause some DNA lesions. In order to avoid deleterious mutations, blockage of replication and transcription, and chromosomal breakage, these lesions need to be repaired. The fact that failure to repair damaged DNA increases the possibility of developing tumors and other diseases highlights the importance of DNA repair. Homologous recombination (HR), the exchange of genetic information between allelic sequences, is a mechanism that repairs a variety of DNA lesions, including double-strand DNA breaks (DSBs), single-strand DNA gaps and interstrand crosslinks. HR is important both for repairing DNA lesions in mitosis and for chromosomal pairing and exchange during meiosis. However, sometimes some forms of HR can also lead to some undesirable DNA arrangements. Therefore, multiple regulatory mechanisms have evolved to ensure that HR takes place at the right time, place and manner.
Homologous recombination pathway and its regulation
The strand-exchange protein, called Rad51 is the central player in HR in eukaryotic cells (RecA in E. coli). Rad51 functions in all three phases of HR, presynapsis, synapsis, and post-synapsis. In the first phase, Rad51 is loaded onto single-strand DNA (ssDNA) which either is generated by degrading 5’-strands at DSBs or arises from replication perturbation. The ssDNA within the filament is stretched as much as half the length of B-form dsDNA, which is essential for fast and efficient homology search. During the next phase, the synapsis, Rad51 helps the formation of a physical connection between the invading DNA substrate and homologous duplex DNA template, resulting in the generation of heteroduplex DNA (D-loop). Rad51-dsDNA filaments are formed by accommodating both the invading and donor ssDNA strands within the filaments. Finally, in the last step 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.
Then there will be at least three different routes which can be used once DNA synthesis is initiated. First, 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-Holiday Junction (dHJ), which is then resolved to produce crossover or non-crossover products 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, which represents the synthesis-dependent strand-annealing mode of HR (SDSA). In the third model, the D-loop will assemble into a process called break-induced replication (BIR). All the pathways above require Rad51, with the exception of some forms of BIR. However, DSBs can also be repaired by pathways independent of Rad51. One of these pathways is the single-strand annealing pathways (SSA), during which ssDNA sequences generated during DSB processing contain regions of homology at both side of DSB and can be annealed and ligated. SSA requires other HR proteins, although it doesn’t need Rad51.
The presence of many kinds of Rad51-dependent pathways and other alternative pathways means there exists some regulatory mechanisms which determine the choices of pathways and manner of execution. In order to control the outcome of repair of different types of lesions, many important decisions need to be made, such as whether SSA and BIR pathways are used only when other repair attempts fail, whether both ends of DSBs are used for repair, and how DNA synthesis is initiated and terminated. Regarding the central role of Rad51 in HR, it is certain that much of the regulation on this protein and its regulators can be determined.
One level of HR regulation happens at the interplay between Rad51 and the ssDNA-binding factor, replication protein A (RPA) complex. RPA has higher affinity for ssDNA than Ras51, therefore, the presence of RPA on ssDNA prevents Rad51 from binding in vitro, which means that RPA-ssDNA formation precedes Rad51 presynaptic filament formation. On the other hand, RPA also promotes recombination by removing secondary structures formed on ssDNA which could impede Rad51 filament formation. In addition, the proteins which can counterpart the inhibitory effect of RPA on Rad51 nucleofilament formation are referred to as recombination mediators. Mediators can facilitate Rad51 loading on ssDNA, increasing intrinsic stability of Ras51 presynaptic filament and protecting Rad51 from removal by factors such as helicases. Rad52 can interact with Rad51 and can also bind RPA once the latter coats ssDNA. To recruit and nucleate Rad51 onto RPA-coated DNA, the Rad51-Rad52 interaction is required. Like Rad51, the Rad55 and Rad57 heterodimers exhibit ATPase activity and bind ssDNA; but unlike Rad51, it can’t catalyse the strand-exchange reaction. Rad54 and Rdh54/Tid1, two members of the Snf2/Swi2 family of DNA-dependent ATPases, play multiple roles in regulating Rad51. They function as positive regulators of Ras51 at early stages of recombination by stabilizing presynaptic filaments, stimulating Rad51-mediated strand invasion, and promoting migration of the branch point of D-loops/HJs, though the latter activity has not yet been demonstrated for Rdh54/Tid1. Also, there are some negative regulators. For example, Srs2 is an anti-recombinase that disassembles Rad51 presynaptic filaments. Mph1 and its homologues, Fml proteins in fission yeast and FANCM in humans, are translocases. They share several activities, including disrupting Rad51-coated D-loops and catalyzing branch migration. The regulatory mechanisms governing HR involve not only the aforementioned positive and negative regulator proteins, but also an intricate network of post-translational modifications (PTMs). Genetic studies provide the first clues for the importance of PTMs, particularly phosphorylation and SUMOylation, in HR regulation. For example, HR in yeast and higher eukaryotic cells is diminished severely with the lack of cyclin-dependent kinase (CDK) and the DNA damage checkpoint.
Relations with human diseases
Genetic studies have showed that there is connection between the germline mutations in several HR genes and predisposition to breast/ovarian and other tumors. HR genes’ somatic mutations have also been uncovered. A role for BRCA1 in the end resection step of HR is well supported. There are at least three mechanisms implicated, antagonizing the resection inhibitor 53BP1, promoting resection in the BRCA1-C complex with CtIP, and inhibiting resection in the BRCA1-A complex through AbraxasRAP800. The BRCA1-PALB2-BRCA2 complex and RAD51 paralogs cooperate to load RAD51 onto ssDNA coated with RPA to form the essential recombination intermediate, the RAD51-ssDNA filament. The crucial role of these genes in key steps of HR provides vital clues for understanding the cause of cancer in patients with mutations in these genes and for investigating more effective cancer treatments.