The major interest of our laboratory is the repair of DNA damage by the homologous recombinational repair (HRR) pathway. This pathway is essential for cell survival in all organisms and serves to repair DNA double-strand breaks (DSBs). In humans, HRR is critical for the repair of radiation-induced DSBs in S/G2 and spontaneous DSBs in S-phase, and is indispensible for maintaining genomic stability, limiting mutagenesis and preventing cancer. The products of HRR are gene conversion (i.e. the asymmetric transfer of sequence information) with or without crossing over (i.e. reciprocal exchange of adjacent sequences). However, if misregulated, the fidelity of this generally precise DNA repair pathway can be severely compromised. For example, crossing over between repetitive sequences throughout the genome can lead to gene amplification, deletion and translocation, and gene conversion between two heterozygous alleles can result in loss of heterozygosity. Such genetic alterations have been observed in human disease, and defects in many of the proteins involved in HRR have been shown to promote carcinogenesis. For example, mutations in the HRR-mediator protein BRCA2 lead to cancer, and defects in HRR-regulating DNA helicases, such as the Bloom’s syndrome protein (BLM), also are associated with a cancer phenotype. As a result, the HRR pathway has become a potential target for tumor therapy.
In eukaryotes, the recombinases RAD51 and DMC1 mediate strand invasion and strand pairing during homologous recombination and form multimers on single-stranded DNA (i.e. filaments). DMC1 is expressed in meiotic cells only and functions during meiotic recombination to generate genetic diversity. RAD51 is the major key player and strand transferase in mitotic recombination and facilitates DNA strand break repair by HRR.
Our research in recent years has dealt primarily with characterizing individual genes, and their encoded proteins, that play important roles in HRR in mitotic human cells. One group of five human proteins we have characterized are known as the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3). These proteins assist RAD51 during HRR, and at least one of their functions is during an early stage of HRR, preceding RAD51 filament formation. We have also recently characterized RAD51AP1 (RAD51-Associated Protein 1), a protein that directly interacts with RAD51 and that plays a role at later stages in HRR (i.e. after RAD51 filament formation). We have collaborated with Dr. Patrick Sung’s group at Yale University in elucidating the biochemistry of RAD51AP1 and found that this protein stimulates the activity of RAD51 after the RAD51 filament has been formed. A second human RAD51-interacting protein (RAD51AP2) has also been characterized, but unlike RAD51AP1, which is expressed in both mitotic and meiotic cells, RAD51AP2 is only expressed during meiosis. Under a grant from NASA, we have also been involved in characterizing the role of HRR in the repair of complex DSBs resulting from the types of radiation that astronauts are exposed to.
Wiese, C., E. Dray, T. Groesser, J San Filippo, I. Shi, D.W. Collins, M.S. Tsai, G.J. Williams, B. Rydberg, P. Sung, and D. Schild. Promotion of homologous recombination and genomic stability by RAD51AP1 via RAD51 recombinase enhancement. Mol. Cell 28:482-490, 2007.
Kovalenko, O.V., C. Wiese, and D. Schild. RAD51AP2, a novel vertebrate- and meiotic-specific protein, shares a conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51. Nucleic Acids Res 34: 5081-5092, 2006.
Wiese C., J.M. Hinz, R.S. Tebbs, P.B. Nham, S.S. Urbin, D.W. Collins, L.H. Thompson, and D. Schild. Disparate requirements for the Walker A and B ATPase motifs of human RAD51D in homologous recombination. Nucleic Acids Res. 34: 2833-2843, 2006.
Lio, Y.C., D. Schild, M.A. Brenneman, J.L. Redpath, and D.J. Chen. Human Rad51C deficiency destabilizes XRCC3, impairs recombination and radiosensitizes S/G2-phase cells. J. Biol. Chem. 279: 42313-42320, 2004.
Thompson, L.H. and D. Schild. Recombinational DNA Repair and Human Disease. Mut. Research 509: 49-78, 2002.
Wiese, C., D.W. Collins, J.S. Albala, L.H. Thompson, A. Kronenberg and D. Schild. Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells. Nucleic Acid Res. 30: 1001-1008, 2002.
Thompson, L.H. and D. Schild. Homologous recombinational repair of DNA ensures chromosome stability in mammalian cells . Mut. Research 477: 131-153, 2001.
Schild, D., Y. Lio, D.W. Collins, T. Tsomondo and D.J. Chen. Evidence for simultaneous protein interactions between human RAD51 paralogs. J. Biol. Chem. 275: 16443-16449, 2000.