Assembly of RecA-like recombinases: distinct roles for mediator proteins in mitosis and meiosis.

Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30 degrees C but not 20 degrees C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G(1), consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.

Genomes OnLine Database (GOLD): a monitor of genome projects world-wide.

GOLD is a comprehensive resource for accessing information related to completed and ongoing genome projects world-wide. The database currently provides information on 350 genome projects, of which 48 have been completely sequenced and their analysis published. GOLD was created in 1997 and since April 2000 it has been licensed to Integrated Genomics. The database is freely available through the URL: http://igweb.integratedgenomics.com/GOLD/.

The breast cancer susceptibility gene BRCA1 is required for subnuclear assembly of Rad51 and survival following treatment with the DNA cross-linking agent cisplatin.

Mutations in breast cancer tumor susceptibility genes, BRCA1 and BRCA2, predispose women to early onset breast cancer and other malignancies. The Brca genes are involved in multiple cellular processes in response to DNA damage including checkpoint activation, gene transcription, and DNA repair. Biochemical interaction with the recombinational repair protein Rad51 (Scully, R., Chen, J., Ochs, R. L., Keegan, K., Hoekstra, M., Feunteun, J., and Livingston, D. M. (1997) Cell 90, 425-435), as well as genetic evidence (Moynahan, M. E., Chiu, J. W., Koller, B. H., and Jasin, M. (1999) Mol. Cell 4, 511-518 and Snouwaert, J. N., Gowen, L. C., Latour, A. M., Mohn, A. R., Xiao, A., DiBiase, L., and Koller, B. H. (1999) Oncogene 18, 7900-7907), demonstrates that Brca1 is involved in recombinational repair of DNA double strand breaks. Using isogenic Brca1(+/+) and brca1(-/-) mouse embryonic stem (ES) cell lines, we investigated the role of Brca1 in the cellular response to two different categories of DNA damage: x-ray induced damage and cross-linking damage caused by the chemotherapeutic agent, cisplatinum. Immunoflourescence studies with normal and brca1(-/-) mutant mouse ES cell lines indicate that Brca1 promotes assembly of subnuclear Rad51 foci following both types of DNA damage. These foci are likely to be oligomeric complexes of Rad51 engaged in repair of DNA lesions or in processes that allow cells to tolerate such lesions during DNA replication. Clonogenic assays show that brca1(-/-) mutants are 5-fold more sensitive to cisplatinum compared with wild-type cells. Our studies suggest that Brca1 contributes to damage repair and/or tolerance by promoting assembly of Rad51. This function appears to be shared with Brca2.

Xrcc3 is required for assembly of Rad51 complexes in vivo.

Rad51 is a member of a family of eukaryotic proteins related to the bacterial recombinational repair protein RecA. Rad51 protein localizes to multiple subnuclear foci in Chinese hamster ovary cells. Subnuclear Rad51 foci are induced by ionizing radiation or the DNA cross-linking agent cisplatin. Formation of these foci is likely to reflect assembly of a multimeric form of Rad51 that promotes DNA repair. Formation of damage-induced Rad51 foci does not occur in the Chinese hamster ovary cell line irs1SF, which is sensitive to DNA damaging agents. The Rad51 focus formation defect of irs1SF cells is corrected by a construct that encodes the repair protein Xrcc3. Xrcc3 is a human homolog of Rad51 previously isolated by virtue of its ability to correct the radiation sensitivity of irs1SF cells. Changes in the steady state level of Rad51 protein do not account for the irs1SF defect nor do they account for the appearance of foci following DNA damage. These results suggest that Xrcc3 is required for the assembly or stabilization of a multimeric form of Rad51 during DNA repair. Cell lines defective in two different components of DNA protein kinase formed Rad51 foci in response to damage, indicating DNA protein kinase is not required for damaged-induced mobilization of Rad51.