Advisor: Dr. Steven James
In the filamentous fungus Aspergillus nidulans, snoARif1 (suppressor-of-nimO) and nimODbf4 (never-in-mitosis) are involved in DNA replication, DNA damage responses (DDR), and the induction and repair of programmed double-strand breaks (DSBs) during meiosis. nimODbf4 encodes the regulatory subunit of nimO-cdc7 kinase, whose ortholog, Dbf4-dependent kinase (DDK), is a highly conserved serine-threonine kinase. Rif1 in budding yeast localizes to telomeres, where it maintains telomere homeostasis by inhibiting both elongation and resection. In mammals, however, Rif1 has evolved new functions, acting in the intra-S phase checkpoint, facilitating recovery from replication stress, and regulating origin firing during DNA replication. In Aspergillus, loss of snoARif1 suppresses the temperature and DNA damage sensitivity of nimODbf4 mutants, suggesting that snoARif1 may act normally to inhibit the role of nimODbf4 in DDR. snoARif1 and nimODbf4 physically interact at a serine-proline rich region of snoARif1, in which are found three conserved SSPP/SSPT tetrapeptide motifs. Substitution of non-phosphorylatable alanine at all six serines (AA6x) confers strong sensitivity to double strand breaks, indicating that these three tetrapeptides in snoARif1 may function as an on/off switch controlling a DNA damage response during mitotic growth. In this study, we have used site-directed mutagenesis to dissect the function of each of these three motifs, by e.g., mutating SSPT to AAPT. Our results demonstrate that, by itself, the second motif may act as the regulatory switch for DNA damage responses. Whereas mutations in the first and third motifs confer only weak sensitivity to genotoxic agents, alanine replacements in the second motif confer strong sensitivity similar to the original AA6x mutant. Experiments are now underway to analyze the role of each individual serine (e.g., ASPT, SAPT) in controlling the response to DNA injury.