The finding of synthetic lethality between Rad52 and BRCA2 opened a significant area of research for the laboratory. We have recently shown that BRCA1 and PALB2 are also synthetic lethal with Rad52, implying that the relationship applies to the whole BRCA1-BRCA2 pathway. These observations allow us to investigate other players in this pathway and to determine epistasis and synthetic lethality with Rad52. In addition, Rad52 is a potential target for therapy in BRCA-deficient tumors (see Translational Research).
How Rad52 is engaged in homologous recombination and how it differs from the recruitment of BRCA1-BRCA2 is under investigation. Rad52 is predominantly recruited in S-phase of the cell cycle, whereas BRCA1-BRCA2 can be engaged after damage more extensively through the cell cycle. In vitro, human Rad52 does not support strand exchange reaction like yeast Rad52, so there is an open question about how human Rad52 mediates Rad51 function. There are links between this project and the phosphorylation of RPA2, which primes the homologous recombination machinery. This leads to the question: Is phosphorylation driving both mediators of Rad51?
The Rad51 paralogs are important for homologous recombination (HR) and the maintenance of genomic stability. Although the role of Rad51 as a recombinase has been well characterized, the molecular mechanism by which the five Rad51 paralogs regulate HR and genomic integrity remains unclear. Biochemically, the Rad51 paralogs associate with one another in two distinct complexes: Rad51B-Rad51C-Rad51D-XRCC2 (BCDX2) and Rad51C-XRCC3 (CX3). We find that the BCDX2 complex is epistatic with the BRCA2 pathway of HR and acts downstream of BRCA2 recruitment, but upstream of Rad51 recruitment to damage sites. We are currently investigating the mechanism by which the BCDX2 complex functions in the BRCA2-dependent pathway.
Site-Specific Replication Block
Many cancer-initiating genetic abnormalities arise from DNA replication encountering DNA damage. If the DNA damage has affected the continuity of the template strand, replication can collapse, producing a one-ended double-strand break. The role of BRCA1 and BRCA2 has been studied in artificially induced DSB in mammalian cells, but their role in repairing collapsed replication forks is unclear. The study of replication block, cleavage, and repair is hampered by a lack of methods for inducible site-specific replication block in mammalian cells. We have developed a novel site-specific approach to inducing fork stalls in mammalian cells by using a process in bacteria in which a protein called Tus can bind a 23-bp terminator (Ter) sequence to induce a replication fork block. This Tus-Ter system will allow us to demonstrate localization of repair proteins, such as BRCA1 and BRCA2, at sites of replication fork stalls using standard biochemical and imaging techniques. To demonstrate the requirement of BRCA1 and BRCA2 in replication-linked repair, the Tus-Ter system will also be used to create a functional assay that will measure fork-stall-induced repair.
Double-Strand-Break-Repair Pathway Choice
DNA double-strand breaks (DSB) may be induced by many endogenous and exogenous factors, such as replication errors, ionizing radiation, and many chemotherapeutic agents. Within the cell there are two main DSB repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). During the G1 phase of the cell cycle, it is predominantly the error-prone NHEJ pathway that is utilized to repair DSBs; however during S/G2 there is a significant contribution from HR as the sister chromatid becomes available for repair. This leads to error-free, faithful repair of the genome, which is vital, as genomic instability may result in tumorigenesis. Despite the apparent clear roles for HR and NHEJ within the cell cycle, the choice of pathway when both are available to the cell is unknown. There is increasing evidence for interaction between the HR proteins and those involved in Fanconi anemia (FA). FA acts upstream of HR during interstrand crosslink repair and coordinates recruitment of HR proteins at the damage site. In those studies where FA proteins have been depleted, there is contradictory evidence as to whether levels of repair via NHEJ or HR are affected. The effect of FA proteins in pathway choice for DSB repair is under investigation. We are employing a double-fluorescence reporter system in cells that have been depleted of various FA proteins using siRNA and flow cytometry as a readout of repair levels.