Repair of DNA damage is essential to preventing mutations and chromosomal rearrangements. The DNA repair process involves many proteins working together in a tightly regulated system to fix the damage. Double-strand breaks (DSBs) are one of the most lethal types of DNA damage, and can arise from both endogenous (i.e. replicative damage or reactive oxygen species) and exogenous sources (i.e. radon). Cells must repair approximately 50 DSBs per day, which correlates with a frequency of one DSB per 108
bp per cell cycle [1
]. Mutations in genes important for DSB repair have been implicated in many cancer predisposition diseases such as ataxia telangiectasia, Nijmegen breakage syndrome, and Bloom syndrome [2
]. The proteins required for DSB repair are highly conserved in eukaryotes from yeast to humans, which highlight the importance of DNA repair throughout evolution. Consequently, cells have evolved many different pathways in the repair of DSBs. For example, the DNA ends can be re-ligated together during non-homologous end joining (NHEJ; ) and micro-homology mediated end joining or, alternatively, a homologous template could be used for repair as in homologous recombination (HR; )[3
Figure 1 Summary of the major double-strand break (DSB) repair pathways. A. Non-homologous end joining (NHEJ) is the simplest form of DNA DSB repair, because it does not require a homologous template. Broken ends are religated together. The advantages of this (more ...)
How does a cell decide which repair pathway to utilize? This has been the topic of much investigation, and recently some pieces of the puzzle have been illuminated by the discovery of novel regulators of the DNA repair pathways. During HR, when a DSB is induced in a cell, the DNA ends are resected and replication protein A (RPA) binds the single-stranded DNA (ssDNA) overhangs that are produced and serves as a general marker for ssDNA in a cell ()[4
]. The multimeric RPA filaments on the ssDNA also serve to protect the unstable ssDNA from further damage. In order for HR to occur, the recombinase protein Rad51 must displace RPA on the ssDNA and form its own filaments. This process is facilitated, in part, by Rad52 in yeast [6
], and BRCA2 and RAD52 in humans ()[7
]. In mammalian cells, the five RAD51 paralogues (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) are required for RAD51 focus formation [10
]. Formation of Rad51 filaments is in essence the crux of the DSB repair pathway, because the Rad51 nucleo-protein filament is essential for all subsequent homology search and strand invasion steps of HR.
Figure 2 Regulation of homologous recombination in yeast and humans. A,B. A double-strand break is induced. Some of the most common causes of DSBs include radon, radiation, reactive oxygen species, cisplatin, etc. C. After DNA end resection, the 3′ overhangs (more ...)
After the DNA ends are resected and Rad51 filaments are formed, a cell is committed to perform HR in order to repair the damaged DNA template (Reviewed in [4
]). Rad51 mediates the search for the homologous DNA sequence and, once the homologous sequence is found, Rad51 filaments facilitate the invasion of the ssDNA overhang into the homologous double-stranded DNA (dsDNA) sequence ( and ). Thus, one strand of the duplex DNA is displaced leaving the complementary strand to serve as a template for repair. This specific recombination structure is referred to as a displacement-loop (D-loop; ). The step in the HR pathway when the D-loop is formed is referred to as a synapsis; consequently, the homologous recombination steps that occur before D-loop formation are referred to as pre-synaptic whereas the latter steps are post-synaptic. The invading end of the D-loop can be extended by the DNA polymerase, which would then copy any information that might be missing at the breaksite. Resolution of the D-loop structure can occur by two different mechanisms. The invading strand of the DNA can be displaced and reanneal to the other broken chromosome end in a process called synthesis dependent strand annealing (SDSA), which leads to only non-crossover products (). Alternatively, the second end of the DSB can be captured, giving rise to a structure called a double-Holliday junction (dHJ; ). Resolution of the dHJs can result in a crossover or non-crossover product (), with the non-crossover product being favored in mammalian somatic cells. Too much or too little HR can be toxic to a cell. For example, a cell that undergoes too much HR is defined as having a hyper-recombinant phenotype. This can result in gross chromosomal rearrangements including duplications, deletions, and translocations [13
]. On the other hand, homologous recombination has the highest fidelity in the repair of dsDNA damage because it utilizes a homologous template. HR is also needed for proper chromosome segregation during meiosis. Therefore, too little HR can lead to an accumulation of mutations [3
]. In this review, we will focus on the key regulators of Rad51 filament formation through evolution from yeast to humans.