From yeast to mammals: recent advances in genetic control of homologous recombination

Abstract

Misregulation of DNA repair is associated with genetic instability and tumorigenesis. To preserve the integrity of the genome, eukaryotic cells have evolved extremely intricate mechanisms for repairing DNA damage. One type of DNA lesion is a double-strand break (DSB), which is highly toxic when unrepaired. Repair of DSBs can occur through multiple mechanisms. Aside from religating the DNA ends, a homologous template can be used for repair in a process called homologous recombination (HR). One key step in committing to HR is the formation of Rad51 filaments, which perform the homology search and strand invasion steps. In S. cerevisiae, Srs2 is a key regulator of Rad51 filament formation and disassembly. In this review, we highlight potential candidates of Srs2 orthologues in human cells, and we discuss recent advances in understanding how Srs2's so-called "anti-recombinase" activity is regulated.

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 process are that it is quick and efficient. The disadvantage is that it is a potentially error-prone repair mechanism since no template is used for repair and genetic information can be lost at the breaksite. B. Synthesis dependent strand annealing (SDSA) is a form of non-crossover homologous recombination, in that it uses a homologous sequence as a template for DNA repair. However, this process does not involve second end capture of the DNA end. After end processing one strand is synthesized using a homologous template and then is re-ligated to the broken end. The newly synthesized strand is then used as a template and base pairs to the complimentary sequence, consequently resolving the break. C. Homologous recombination uses a homologous template for repair. After the DNA ends are resected, the ssDNA 3′ overhang invades a homologous sequence and restores any missing information at the break site. The second end of the DNA is captured resulting in a double-Holliday junction that can be resolved into a crossover or non-crossover product depending upon where the junctions are cut. Legend: Red-Blue double-helix is the broken molecule of DNA. Grey DNA is the homologous sequence. Blue highlighted segments of DNA are newly synthesized pieces. Figure is not to scale.

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 are coated with RPA, which forms a filament and is a general marker for ssDNA in the cell. D. Replacing RPA with RAD51 is an important step in initiating HR. In yeast Rad52, as well as Rad55-Rad57, helps load Rad51 onto ssDNA thus promoting HR. In humans, RAD52 and BRCA2 function as positive regulators of HR by facilitating the disassembly of RPA filaments and the nucleation and expansion of RAD51 filaments. Both pre-synaptic Rad51 regulation (D.) and D-loop disassembly (E.) is mediated by Srs2 in yeast. Srs2 is negatively regulated by both the Shu complex and the Rad55-Rad57 heterodimer. In humans presynaptic regulation of RAD51 is accomplished by RECQL5 and PARI. Both of these proteins interact with PCNA, but how they are regulated has yet to be fully elucidated. The next step after Rad51 filament formation is the homology search, which requires less than 16 bp for pairing and 80 bp for strand exchange [72]. E. The disassembly of the D-loop is another way to regulate homologous recombination. In yeast, D-loop disassembly is mediated by Srs2 while in humans, RTEL performs an analogous function. Figure is not to scale.

Figure 3

Proposed mechanisms of Srs2 and PARI. PARI has many similarities to the yeast anti-recombinase Srs2, suggesting that PARI is a strong candidate for being a mammalian homologue of Srs2. A. Both Srs2 and PARI are recruited to the replication fork by PCNA where they can regulate illegitimate homologous recombination during DNA synthesis. Both proteins use their Rad51/RAD51 and DNA binding sites to physically interact with Rad51/RAD51 filaments on ssDNA. B. Rad51’s conformation when it is bound to ATP favors filament formation while its ADP bound form favors disassociation. The direct interaction with Rad51/RAD51 by Srs2 and PARI stimulates Rad51’s intrinsic ATP hydrolyzing activity and the disassociation of one monomer of Rad51 from the filament. C. Srs2 uses its helicase activity to move down ssDNA to interact with the next unit of Rad51, thus perpetuating filament disassembly. On the other hand, PARI lacks Walker A and Walker B motifs, and therefore it cannot hydrolyze ATP on its own. Without an active helicase functionality, it is unclear how PARI can move down ssDNA and facilitate the disassembly of additional RAD51 monomers from the filament. Figure not to scale.