Abstract 1937 Intrinsic disorder in the C-terminal Domain of the Yeast Srs2 helicase Provides Structural Diversity Essential to Implementing Multi-functional Roles in replication, Recombination and Repair

The Srs2 helicase from Saccharomyces cerevisiae acts as an ATP-dependent translocase and helicase in primarily the 3'-5' direction and as an anti-recombinase through direct interaction between the C-terminal domain (CTD) and Rad51 protein. It promotes gap repair, replication-restart and translesion DNA synthesis through interactions with Exo1 nuclease and PCNA-SUMO, respectively, while deterring Rad51 protein filament formation. The N-terminal 776 residues (1174 total) share structural and functional homology with other SF-1 helicases such as UvrD and Rep. The SRS2 CTD, which shares little homology to other helicases, comprises the remaining ∼400 amino acids. Aside from Rad51 protein, over 166 different proteins potentially interact with the SRS2 CTD, and it harbors multiple CDK-1 consensus phosphorylation and SUMO modification sites. The CTD is not required for DNA helicase or translocase activity but is essential for anti-recombinase activity and Srs2 regulation. Evidence suggests that phosphorylation at one or more sites in the CTD or different protein interactions modulates Srs2 activity and role in response to cellular signals. However, the essential structural characteristics and conformations of the Srs2 CTD remain unknown except for a crystallized complex between a small CTD fragment from its end and PCNA-SUMO. The extent or instance(s) of phosphorylation at CTD sites have also not been correlated with changes in Srs2 activity or conformational forms. To address this gap in knowledge, we applied computational methods to analyze the amino acid composition and sequence of the Srs2 CTD and the predicted pattern and frequency of modification at consensus CDK-1 phosphorylation sites. Our results suggest that the Srs2 CTD is intrinsically disordered due to features shared with known intrinsically disordered proteins (IDPs) or protein regions (IDRs); these proteins tend to sample many conformations of limited stability over time with conformation frequency and lifetime shifted by modifications or protein interactions. Further computational analysis with disorder prediction software confirms the presence of significant disorder within the CTD ranging amino acids 850- 1030. We have preliminary results from molecular dynamics simulations focused on the Srs2 CTD (783-1174) using GROMACS with input files generated by I-TASSER and CHARMM-GUI under biological conditions. Results are consistent with active motion and instability of the CTD over the entire time-period sampled. Additional simulations will compare differences with SeràGlu mutations at consensus phosphorylation sites at amino acid positions, 879, 893, 938, 950, 965. The Srs2 CTD (783-1174) has also been expressed in E.coli and purified along with four CTD phosphomimetic mutants (Ser to Glu) at single and multiple sites. Initial results with fluorescence quenching of the wild-type CTD fragment show extensive exposure of the single trp (812) to quencher agents consistent with IDR properties.