Project Grant R35GM155148

FORMATION AND REPAIR MECHANISMS OF DNA-PROTEIN CROSS-LINKS, AND FUNCTIONS AND REGULATORY MECHANISMS OF TREX1 IN DNA REPAIR - ABSTRACT UNDERSTANDING HOW DNA IS DAMAGED AND HOW THE DAMAGE IS REPAIRED IS CRITICAL. THIS IS BECAUSE DNA DAMAGE CONTRIBUTES TO GENOME INSTABILITY, AGING, AND DISEASES, AND DNA-DAMAGING AGENTS ARE USED IN CHEMOTHERAPY. COVALENT DNA-PROTEIN CROSS-LINKS (DPCS) ARE UBIQUITOUS AND BULKY DNA LESIONS. DESPITE THAT IT HAS BEEN WELL ACCEPTED THAT DPCS ARE HIGHLY TOXIC, COMPARED TO OTHER TYPES OF DNA DAMAGE, DPCS ARE MUCH LESS WELL STUDIED MAINLY DUE TO THE LACK OF APPROACHES TO DETECT, QUANTIFY, AND SYNTHESIZE DPCS. MY RESEARCH PROGRAM STUDIES DPCS AT 3-DNA TERMINI (3-DPCS) WITHIN SINGLE-STRAND BREAKS (SSBS). THEY ARE DERIVED FROM THE APURINIC/APYRIMIDINIC (AP) SITE THAT IS ONE OF THE MOST FREQUENTLY FORMED DNA LESIONS AND INDUCED BY MANY ENDOGENOUS AND EXOGENOUS GENOTOXINS INCLUDING SOME ANTI-CANCER DRUGS. IF LEFT UNREPAIRED, 3-DPCS WILL BLOCK DNA REPLICATION AND TRANSCRIPTION, PREVENT THE SSB REPAIR, CAUSE GENOME INSTABILITY, AND MAY LEAD TO CELL DEATH. WE HYPOTHESIZE THAT 3-DPC FORMATION IS A PREVIOUSLY UNCHARACTERIZED CYTOTOXIC MECHANISM OF THE AP SITE AND ITS INDUCING AGENTS, 3-DPCS ARE NEW BIOMARKERS OF OXIDATIVE STRESS-RELATED DISEASES, AND INHIBITING 3-DPC REPAIR WILL SYNERGIZE DNA METHYLATING AGENTS. OUR GOAL IS TO ELUCIDATE THE FORMATION AND REPAIR MECHANISMS OF 3-DPCS. IN PAST WORK, WE HAVE DETECTED 3-DPCS IN HUMAN CELLS USING A NOVEL MASS SPECTROMETRY PIPELINE, BUT THEIR CELLULAR ABUNDANCE AND TO WHAT EXTENT THEY ARE INDUCED BY GENOTOXINS ARE UNKNOWN. WE HAVE CHEMICALLY SYNTHESIZED 3-DPCS AND DEMONSTRATED THAT THEY CAN BE REPAIRED BY THREE HUMAN NUCLEASES, BUT ONLY WHEN THE CROSS-LINKED PROTEINS ARE INITIALLY DIGESTED BY TRYPSIN. HOW 3-DPCS ARE PROTEOLYZED IN CELLS REMAINS ELUSIVE. WE WILL FILL THESE KNOWLEDGE GAPS. NOTABLY, DURING STUDYING 3-DPC REPAIR, WE DISCOVERED IN VITRO THAT PROTEOLYZED 3- DPCS AND AN AP SITE REPAIR INTERMEDIATE (I.E., 3-PUA) ARE EXCISED BY HUMAN THREE-PRIME REPAIR EXONUCLEASE 1 (TREX1). THIS UNEXPECTED FINDING IS THE FIRST TO REPORT A DIRECT ROLE OF TREX1 IN DNA REPAIR AND CHALLENGES THE PREVIOUS NOTION. IT OPENED A NEW AND EXCITING RESEARCH DIRECTION THAT WE WILL ALSO PURSUE IN THIS MIRA APPLICATION. OUR HYPOTHESIS IS THAT TREX1 IS A 3-DNA LESION PROCESSING ENZYME AND A PROMISING THERAPEUTIC TARGET. OUR GOAL IS TO DELINEATE THE FUNCTIONS AND REGULATORY MECHANISMS OF TREX1 IN DNA REPAIR. WE WILL FOCUS ON SEVERAL IMPORTANT QUESTIONS. FOR INSTANCE, DOES TREX1 PLAY A ROLE IN CELLS IN RESPONSE TO DNA DAMAGE? IF SO, IS THAT DEPENDENT ON ITS EXONUCLEASE ACTIVITY? AND HOW IS TREX1 RECRUITED AND REGULATED? WE WILL ADDRESS THESE QUESTIONS AND STUDY 3-DPC FORMATION AND REPAIR MECHANISMS USING INTERDISCIPLINARY TECHNIQUES INCLUDING ORGANIC SYNTHESIS, QUANTITATIVE MASS SPECTROMETRY, PROTEOMICS, BIOCHEMISTRY, MOLECULAR AND CELL BIOLOGY-BASED APPROACHES. THIS RESEARCH WILL ADVANCE FUNDAMENTAL UNDERSTANDING OF DNA DAMAGE AND REPAIR. SUCH NEW KNOWLEDGE WILL INFORM THE DEVELOPMENT OF NOVEL THERAPEUTIC INTERVENTIONS.