Abstract 1129 Examination of the Biochemical Mechanism of SPRTN in the Selection and Removal of DNA-Protein Crosslinks

SPRTN (Spartan) is a mammalian protease that selectively targets and repairs DNA-protein crosslinks (DPCs). DPCs are one of the most ubiquitous lesions that form as proteins covalently bind to DNA, through a variety of physical and chemical exposures. An unresolved accumulation of DPCs, which forms from the depletion of SPRTN enzymatic activity, is known to cause genomic instability. This instability, in patients, most commonly manifests as Ruijs-Aalfs syndrome and can later evolve into hepatocellular carcinoma. However, the primary step to treatment is uncovering the mechanism of action of the SPRTN active site. In this procedure, our first aim is to introduce four different point mutations within the SPRTN Nudix domain, via site-directed mutagenesis. Once the mutant library is complete, the recombinant SPRTN mutants will be expressed through E. Coli and purified through GST (Glutathione-S-transferase) affinity chromatography. The mutants' enzymatic activity will be examined through in vitro proteolysis. Conclusions drawn from the SPRTN mutants' enzymatic activity will provide further insight into the mechanism of proteolysis within the SPRTN domain which could aid in the creation of a novel treatment for Ruijs-Aalfs syndrome. In this study, a successful mutation within the SPRTN Nudix domain through site-directed mutagenesis is yet to appear. Therefore, the enzymatic activity of the SPRTN protease has not yet been fully examined. However, the hypothesis remains to be that DPCs are tagged through post-translational modification and targeted for proteolytic breakdown by SPRTN. Freshman Research Initiative, College of Natural Sciences, The University of Texas at Austin.