Structure function studies of Sml1, an inhibitor of DNA : synthesis in Saccharomyces cerevisiae

Smll is a protein found in yeast that is implicated in regulating ribonucleotide reductase (RNR) and is a member of the Rad53/Mecl cell cycle checkpoint pathway. RNR catalyzes the reduction of ribonucleotides to deoxyribonucleotides. This action is the ratelimiting step of DNA precursor synthesis. 5ml 1 has been shown in other studies to bind to Rnrl, of yeast, thereby inhibiting the reduction activity when DNA synthesis is not required (Zhao, X., Mueller, EGO., and Rothstein, R. ( 1998)). The project presented here utilizes recombinant, truncated mutant forms of Smll and site-directed mutants to investigate the oligomerization and phosphorylation of Smll. Wild type Smll has been truncated by 8, 20, and 38 amino acids, omitting the N-terminal region, which is not thought to interact with Rnrl. These mutants have been used to investigate the oligomeric state of Smll by size-exclusion chromatography and sedimentation equilibrium analytical ultracentrifugation. To characterize phosphorylation of 5ml 1 several serines have been mutated to alanines and a glutamic acid was mutated to a glutamine. The results from these studies have led to the identification of the phosphorylation sites and the key recognition residues of Smll necessary for Dunl kinase activity.