Project Grant F31CA314258

THE ROLE OF RAD52'S INTRINSICALLY DISORDERED C-TERMINAL DOMAIN IN ALT CANCERS - PROJECT SUMMARY TELOMERES ARE PROTECTIVE DNA-PROTEIN STRUCTURES AT CHROMOSOME ENDS THAT SHORTEN WITH EACH CELL DIVISION, ULTIMATELY TRIGGERING SENESCENCE OR APOPTOSIS. TO BYPASS THIS LIMIT, CANCER CELLS REACTIVATE TELOMERE ELONGATION MECHANISMS. WHILE MOST TUMORS RELY ON TELOMERASE, APPROXIMATELY 15% USE AN ALTERNATIVE LENGTHENING OF TELOMERES (ALT) PATHWAY, A HOMOLOGY-DIRECTED REPAIR MECHANISM. ALT- POSITIVE CANCERS, INCLUDING OSTEOSARCOMAS AND GLIOBLASTOMAS, ARE AGGRESSIVE AND LACK TARGETED THERAPIES, UNDERSCORING THE NEED TO UNDERSTAND ALT BIOLOGY AND IDENTIFY THERAPEUTIC VULNERABILITIES. RAD52 IS A DNA REPAIR PROTEIN IMPLICATED IN ALT, YET ITS C-TERMINAL DOMAIN (CTD) REMAINS POORLY CHARACTERIZED. THE CTD LACKS ENZYMATIC ACTIVITY AND DIRECT DNA-BINDING CAPABILITY BUT CONTAINS INTERACTION SITES FOR RAD51 AND RPA AND IS PREDICTED TO BE INTRINSICALLY DISORDERED. RECENT EVIDENCE SUGGESTS RAD52 UNDERGOES PHASE SEPARATION, FORMING CONDENSATES THAT ORGANIZE REPAIR FACTORS AT ALT TELOMERES. THE CTD MAY DRIVE THIS BEHAVIOR, BUT ITS MOLECULAR CONTRIBUTIONS ARE UNKNOWN. EMERGING DATA SUGGEST RAD52 IS POST-TRANSLATIONALLY MODIFIED BY SUMO, A SMALL UBIQUITIN- LIKE MODIFIER THAT FACILITATES PROTEIN-PROTEIN INTERACTIONS. OUR LAB HAS SHOWN THAT SUMO FUSION OF RAD52 ENHANCES ALT PHENOTYPES, AND BIOINFORMATIC ANALYSES PREDICT RAD52 SUMOYLATION SITES WITHIN THE CTD. HOWEVER, THE FUNCTIONAL IMPACT OF CTD SUMOYLATION ON RAD52'S ROLE IN ALT REMAINS UNEXPLORED. ADDITIONALLY, IN VITRO STUDIES REVEAL RAD52 CTD BINDS RNA WITH MICROMOLAR AFFINITY. TERRA, A TELOMERIC NONCODING RNA ENRICHED AT ALT TELOMERES, PLAYS AN IMPORTANT ROLE IN INDITIATING THE ALT PATHWAY BY FORMING RNA:DNA HYBRIDS TO CAUSE REPLICATION STRESS. TERRA ALSO IS REPORTED TO WHETHER THE CTD-TERRA INTERACTS AND HOW THIS INTERACTION CONTRIBUTES TO ALT ARE UNKNOWN. THIS PROPOSAL AIMS TO DEFINE THE MOLECULAR MECHANISMS BY WHICH RAD52 CTD CONTRIBUTES TO ALT TELOMERE MAINTENANCE. AIM 1 WILL INVESTIGATE HOW CTD SUMOYLATION MODULATES RAD52'S PHASE SEPARATION AND INTERACTIONS WITH DNA REPAIR FACTORS. AIM 2 WILL ASSESS RAD52 CTD'S CAPACITY TO BIND TERRA AND ITS ROLE IN RNA-MEDIATED ALT ACTIVITY. WE WILL COMBINE CLASSIC BIOCHEMICAL AND CELL BIOLOGICAL TOOLS WITH PHASE SEPARATION ASSAYS AND THE STATE-OF-THE-ART CHEMICAL INDUCERS OF PROTEIN DIMERIZATION SYSTEMS. BY USING THE BIOPHYSICAL FRAMEWORK OF PHASE SEPARATION AND EMPLOYING AN ARRAY OF INTERDISCIPLINARY TOOLS, WE WILL ELUCIDATE RAD52 CTD-SPECIFIC FUNCTIONS. THE RESULTS WILL ADVANCE OUR UNDERSTANDING OF ALT REGULATION AND INFORM THERAPEUTIC STRATEGIES TARGETING RAD52 IN ALT-POSITIVE CANCERS.