Jeffrey Moyer
Advisor: Dr. Steven James

The universally conserved MRN complex (Mre11-Rad50-Nbs1) is essential for early steps in the repair of DNA damage in all eukaryotes.  The Aspergillus nidulans ortholog of Nbs1is scaA^Nbs1 and is the focus of this study.  scaA^Nbs1 plays a key role in the initial steps of DNA damage repair by activating the MRN complex.  Based on our observation that scaA^Nbs1 interacts genetically with a novel DSB repair protein, snoA^Rif1 (suppressor-of-nimO), we wished to create a set of tools for microscopic localization and biochemical analysis of the scaA protein.  To accomplish this, I inserted the green fluorescent protein (GFP), red fluorescent protein (mRFP), and S-peptide (S-tag) at the carboxyl-terminus of the scaA gene via one-step gene replacement.  The 3-way tagging constructs were synthesized and transformed into two strains of A. nidulans, and correct integration was verified by trans-locus PCR.  In future experiments we wish to use fluorescence microscopy to study the cellular dynamics of tagged scaA protein in response to DNA damage and especially in cells harboring defects in snoA.  Crosses are currently underway to combine, for example, scaA::mRFP with GFP tagged mutations in snoA, and other genes of interest.  (Supported by Senior Project Fund award from the Gettysburg College Provost office)