Louise Juhl:
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Homologous recombination (HR) provides a mechanism to restore integrity and maintain stability of the genetic material. HR is a major pathway for repair of DNA double-strand breaks (DSB), recovery of broken replication forks and generation of meiotic crossovers. The defining step in HR is homologous strand exchange directed by the RecA-related recombinase Rad51. BRCA2 participates in HR by mediating Rad51 homology-directed repair. Both BRCA2 and Rad51 are essential for HR, DNA repair, and the maintenance of genome stability. In the present study, we seek to understand the mechanism of BRCA2 in governing the activity of Rad51 and to learn how other recombination-associated proteins such as DSS1 and RAD52 contribute to its regulation.
We use the yeast-like fungus Ustilago maydis and the avian DT40 cell line as experimental systems since both have a well-conserved BRCA2-based recombinational repair system that resembles the one seen in human. In U. maydis, we show that Brh2, the BRCA2 homologue, and Dss1 colocalize at DNA damage-induced foci, with Dss1 exhibiting a dynamic association with Brh2 foci. Dss1 focus formation is dependent on interaction with full-length Brh2, and the Dss1-Brh2 interaction is required for resistance to DNA damage. In avian DT40 cells, we show that endogenously tagged DSS1 redistributes into subnuclear foci in response to DNA damaging agents. However, DSS1 rarely colocalizes with BRCA2. Our data also indicate that both U. maydis Dss1 and avian DSS1 are involved with the proteasome.
In U. maydis, the Rad52 orthologue seems relegated to a less prominent role than in Saccharomyces cerevisiae, similar to what is found in human cells. We find that Rad52 functionally overlaps with Brh2-ΔNBD, and Rad52 colocalizes with both Brh2 and Rfa1 (subunit of RPA) in response to DNA damage, suggesting a central role for Rad52 in HR. Our data indicate that Brh2 and Rad52 may also function in two redundant pathways. In avian DT40 cells, RAD52 colocalizes with BRCA2/RPA and TopBP1/RPA in response to CPT treatment. In response to UV irradiation, BRCA2 and Polη show colocalization, whereas RAD52 foci do not colocalize with either BRCA2 or Polη. Our data indicate that RAD52 functions together with BRCA2 at a subset of HR events, and RAD52 may cooperate together with BRCA2 in promoting replication fork restart.
Finally, using a RAD52 small molecule inhibitor, we show that we can interfere with RAD52-mediated repair at sites of CPT-induced DNA damage. The synthetic lethality approach using RAD52 small molecule inhibitors in brca-deficient cancers is a promising therapeutic strategy for cancer treatment.