Project Grant 2414612

COLLABORATIVE RESEARCH: ILLUMINATION OF SMC5/6 MANAGEMENT OF DNA REPAIR INTERMEDIATE STRUCTURES -THE MASSIVE AMOUNTS OF GENOMIC DNA IN THE CELL MUST UNDERGO MAJOR STRUCTURAL TRANSFORMATIONS WHEN GENETIC INFORMATION IS REPLICATED OR REPAIRED. DURING THESE PROCESSES, BRANCH-SHAPED DNA STRUCTURES ARE FORMED AS INTERMEDIATES AND THEN ARE PROCESSED INTO LINEAR DNA PRODUCTS. MANY PROTEIN FACTORS COLLABORATE DURING THE FORMATION AND PROCESSING OF DIFFERENT TYPES OF BRANCHED DNA STRUCTURES. HOW MANY OF THESE PROTEINS INTERACT WITH BRANCHED DNAS AND ENABLE THEIR PROCESSING IS NOT CLEAR. THE OBJECTIVE OF THIS COLLABORATIVE PROJECT IS TO DETERMINE HOW A GENOME-PROTECTING COMPLEX CALLED SMC5/6 MANAGES VARIOUS BRANCHED DNA STRUCTURES. THE TWO RESEARCH TEAMS WILL COMBINE BIOCHEMICAL AND BIOPHYSICAL EXPERTISE TO ACHIEVE HIGH-RESOLUTION UNDERSTANDING OF SMC5/6 STRUCTURE AND FUNCTION. THE OUTCOMES WILL ADVANCE KNOWLEDGE OF HOW CELLS QUICKLY RESOLVE DNA REPAIR INTERMEDIATES TO ENSURE GENOME INTEGRITY. IN ADDITION, THE PROJECT WILL HAVE EDUCATIONAL IMPACT THROUGH WORKSHOPS THAT CULTIVATE INTEREST IN DNA AND GENOME RESEARCH AMONG MIDDLE-HIGH SCHOOL STUDENTS AND TEACHERS, AS WELL AS INTERDISCIPLINARY RESEARCH TRAINING OPPORTUNITIES FOR UNDERGRADUATE AND GRADUATE STUDENTS. SMC5/6 IS EMERGING AS AN IMPORTANT AND HIGHLY CONSERVED PROTEIN COMPLEX THAT IS REQUIRED FOR THE PROCESSING OF DNA REPAIR INTERMEDIATES. HOWEVER, THE MECHANISM OF ACTION OF SMC5/6, INCLUDING ITS INTERACTIONS WITH BRANCHED DNA STRUCTURES IS POORLY UNDERSTOOD. THIS STUDY WILL PROVIDE CRITICAL INSIGHTS INTO THE MOLECULAR FUNCTIONS OF SMC5/6. SPECIFICALLY, THE TWO RESEARCH TEAMS WILL EXAMINE HOW SMC5/6 RECOGNIZES HOLLIDAY JUNCTION RECOMBINATIONAL REPAIR INTERMEDIATES AND R-LOOP RNA-DNA HYBRID STRUCTURES, USING COMPLEMENTARY BIOCHEMICAL AND HIGH-SPEED ATOMIC FORCE MICROSCOPY (HS-AFM) METHODOLOGIES. IN ADDITION, HS-AFM IMAGING WILL BE USED TO DIRECTLY OBSERVE THE SEQUENTIAL STEPS WHEREBY DNA HELICASES RESOLVE THESE STRUCTURES AND HOW SMC5/6 AFFECTS THESE STEPS. BY INTEGRATING DYNAMIC HS-AFM DATA WITH BIOCHEMICAL DATA, THE PROJECT HAS POTENTIAL TO PROVIDE NOVEL INSIGHTS INTO DNA REPAIR. GIVEN THE IMPORTANCE OF SMC5/6 IN GENOME MAINTENANCE AND ITS EVOLUTIONARY CONSERVATION IN EUKARYOTES, THIS NEW KNOWLEDGE COULD ILLUMINATE DNA REPAIR MECHANISMS IN DIVERSE ORGANISMS. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.