Mechanism of Smc5/6-mediated protein sumoylation in DNA repair

Genomic DNA is vulnerable to myriad endogenous and exogenous damaging agents, such as ionizing and ultraviolet radiation, chemical carcinogens, and cellular metabolites. Cells have evolved a dozen DNA repair pathways to repair these lesions efficiently and with high fidelity. Amongst these pathways, homologous recombination (HR) is a conserved pathway for the repair of DNA double-strand breaks (DSBs) and damaged replication forks. In eukaryotic cells, HR requires tight regulation to achieve timely activation and suppression. One such regulation relies on sumoylation, a post-translational modification that can control the functions of many substrates including HR proteins. Dysfunction of protein sumoylation in HR often leads to accumulation of HR intermediates, gross chromosomal rearrangements and cancer. Currently, how different sumoylation enzymes confer sumoylation of specific HR proteins at DNA damage sites is poorly understood. Protein sumoylation requires SUMO E3 ligases to enable efficient SUMO transfer from SUMO E2 to substrates. One of the SUMO E3s — the eight-subunit Smc5/6 complex critical to genome maintenance — is a unique composite SUMO ligase. How the SUMO E3 subunit of Smc5/6, Nse2, collaborates with other subunits capable of DNA binding and ATP hydrolysis in substrate sumoylation is poorly understood. In collaboration with Dr. Xiaolan Zhao at MSKCC, we have cellular data suggesting that non-E3 subunits of Smc5/6 collaborate with Nse2 E3 in sumoylation. In this project, we will use biochemical approaches to determine whether DNA binding, ATP binding and hydrolysis activities and SUMO interaction of the subunits of Smc5/6 contribute to efficient sumoylation of Smc5, Smc6 and Sgs1 substrates. We will achieve this with work towards three specific aims: (1) determine how DNA regulates Smc5/6-mediated sumoylation; (2) determine the ATP effects on Smc5/6 sumoylation; (3) examine how Nse2–SIM1, Nse5–SIM and Smc5–SIM affect sumoylation. In Aim 1, we will use our in vitro sumoylation systems with purified holo-Smc5/6 or hexamer Smc5/6 as E3, to address how different types of DNA can influence the sumoylation functions of Smc5/6 (Aim 1a); and to address how Smc5/6 mutant variants defective in each of the known DNA binding sites affect sumoylation (Aim 1b). In Aim 2, we will use two mutants of hexamer Smc5/6, defective of ATP binding and ATP hydrolysis, respectively, to determine the effects of ATP binding and hydrolysis by Smc5/6 on sumoylation. In Aim 3, we will examine the roles of SUMO interaction motifs (SIMs) on Nse2, Smc5 and Nse5 in Smc5/6-mediated sumoylation. We will purify Smc5/6 mutants containing SIM mutations in Nse2, Smc5 and Nse5, and use them to examine how they impact the ability of complex to interact with SUMO in vitro and how they affect in vitro sumoylation. The results from our project will provide insights into the mechanisms of an essential SUMO E3 complex that plays pivotal roles in HR repair and genome stability, and may provide new understanding and therapeutic strategies for human diseases related to dysfunctional sumoylation. 1 Project Number: 1R16GM159631-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: XIAOYU XUE | Institution: TEXAS STATE UNIVERSITY, SAN MARCOS, TX | Award Amount: $148,228 | Activity Code: R16 | Study Section: Special Emphasis Panel[ZGM1 RCB-M (SU)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11198640