Coping with stalled replication forks: investigating the roles of DNA repair genes and checkpoint regulators in the S phase DNA Damage Response

Michael MarllMarll
Advisor - Steven James

We study DNA damage checkpoint and repair pathways that enable cells to survive the stalling of replication forks (RFs) during DNA synthesis (S-phase). We are examining the roles of three checkpoint and DNA repair genes in the fungus Aspergillus nidulans: the Holliday Junction resolvase mus81/eme1 aids in repair by performing structure specific cleavage at collapsed RFs; the checkpoint kinase chkAChk1 recruits several proteins that stabilize stalled RFs; and the DNA synthesis regulator nimODbf4 arrests S-phase progression via a N-terminal checkpoint domain, the BRDF (BRCT and Dbf4-similiarity domain). Double mutants carrying deletions of chkA and mus81 are synthetically lethal with a nimO mutant lacking the BRDF domain (nimO¿BRDF ¿chkA and nimO¿BRDF ¿mus81). This suggests that nimO acts in a pathway distinct from chkA and mus81. Each of these three single mutants enhances sensitivity to double strand break (DSB)-inducing chemicals. We have discovered mutations in a fourth gene, snoARif1 (suppressor of nimO), that reduce the DNA damage sensitivities of nimO¿BRDF and ¿chkA single mutants. To determine whether chkA and mus81 act through different pathways and to identify other pathways that may be influenced by snoA, we are assessing genetic interactions in double mutants carrying, e.g., ¿chkA ¿mus81 and ¿mus81 ¿snoA, as well various triple mutant combinations.