This proposal is for the renewal of a previous grant, which covered the study of the REC1 and REC2 gene products of Ustilago maydis. The Rec2 protein was shown to be a strand-transferase analogous to the RecA gene product of E. coli; study of REC2 and its gene product has been split off from the current proposal, and is covered by its own grant. It will not be discussed further here. The REC1 gene, the sole subject of this proposal, plays multiple roles in DNA repair, mutagenesis, recombination, meiosis, and the control of cell cycle progression in response to DNA damage. It encodes a protein that has in vitro 3' to 5' exonuclease activity and that has weak homology to proofreading exonucleases domains of DNA polymerases, although it is clear that other domains of the protein are required for full in vivo function. Phenotypic analysis of rec1 mutants indicates that the REC1 gene product plays a central role in the maintenance of genome integrity. Null mutants are radiation sensitive and experience elevated mutation rates; the former is most likely due to a failure to arrest at the G2-M boundary following DNA damage, while the latter is consistent with either a defect in a polymerase-coupled proofreader or a defect in post- replicative mismatch repair. Intriguingly, some C-terminal truncations that retain in vitro exonuclease activity are still anti-mutators but have lost the damage-induced checkpoint arrest. This is taken to indicate that the Rec1 protein may be bifunctional. Because of the multiplicity of phenotypes and the apparent bifunctional nature of the protein, Dr. Holloman proposes a research program aimed at determining more precisely the actual function(s) that the REC1 gene product performs. Proper biochemical studies of the exonuclease activity have been hampered by the fact that the primary source of enzyme is protein overexpressed in E. coli, which may be largely inactive or have altered activity. Several strategies are proposed to purify Rec1 expressed in eucaryotic cells, including one using an Ustilago expression system. This protein will be used in vitro to examine substrate specificity and biochemical properties, including possible mismatch- correction activity. The direct role of Rec1 in mismatch correction will be tested using a heteroduplex transformation assay similar to that used fruitfully to study Saccharomyces mismatch repair functions; a simple-repeat instability assay similar to that used successfully in yeast and larger organisms will also be used. The possible bifunctional nature of the REC1 protein will be examined in studies that mutagenize the putative exonuclease and checkpoint regions, to identify regions and residues important for both functions. The effects of these mutations will be examined in both in vitro and in vivo screens. Finally, several strategies are described to identify proteins that interact with Rec1. Preliminary purification data indicate that the protein is part of a multisubunit complex; this finding will be further pursued using the usual set of protein interaction probe tools (i.e. copurification, GST fusions, affinity columns, 2-hybrid system).
The aim i s to identify likely interactors and to isolate mutants in these proteins by the usual reverse genetics approaches. It is anticipated that this may identify other genes that play a role in REC1-dependent activities.
| de Sena-Tomás, Carmen; Yu, Eun Young; Calzada, Arturo et al. (2015) Fungal Ku prevents permanent cell cycle arrest by suppressing DNA damage signaling at telomeres. Nucleic Acids Res 43:2138-51 |
| de Sena-Tomás, Carmen; Sutherland, Jeanette H; Milisavljevic, Mira et al. (2015) LAMMER kinase contributes to genome stability in Ustilago maydis. DNA Repair (Amst) 33:70-7 |
| Zhou, Qingwen; Holloman, William K (2014) Dual DNA-binding domains shape the interaction of Brh2 with DNA. DNA Repair (Amst) 22:104-11 |
| Kojic, Milorad; Sutherland, Jeanette H; Pérez-Martín, José et al. (2013) Initiation of meiotic recombination in Ustilago maydis. Genetics 195:1231-40 |
| Yu, Eun Young; Kojic, Milorad; Holloman, William K et al. (2013) Brh2 and Rad51 promote telomere maintenance in Ustilago maydis, a new model system of DNA repair proteins at telomeres. DNA Repair (Amst) 12:472-9 |
| Kojic, Milorad; Holloman, William K (2012) Brh2 domain function distinguished by differential cellular responses to DNA damage and replication stress. Mol Microbiol 83:351-61 |
| Zhou, Qingwen; Kojic, Milorad; Holloman, William K (2012) Dss1 release activates DNA binding potential in Brh2. Biochemistry 51:9137-46 |
| Holloman, William K (2011) Unraveling the mechanism of BRCA2 in homologous recombination. Nat Struct Mol Biol 18:748-54 |
| Kojic, Milorad; Zhou, Qingwen; Fan, Jie et al. (2011) Mutational analysis of Brh2 reveals requirements for compensating mediator functions. Mol Microbiol 79:180-91 |
| de Sena-Tomás, Carmen; Fernández-Álvarez, Alfonso; Holloman, William K et al. (2011) The DNA damage response signaling cascade regulates proliferation of the phytopathogenic fungus Ustilago maydis in planta. Plant Cell 23:1654-65 |
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