Project Grant F31CA309889

NOVEL MECHANISMS OF GENOMIC INSTABILITY INDUCED BY BENZO[A]PYRENE EXPOSURE DURING CARCINOGENESIS - PROJECT SUMMARY THIS F31 APPLICATION INVESTIGATES A NOVEL MECHANISM OF GENOMIC INSTABILITY INDUCED BY BENZO[A]PYRENE (BAP), A POTENT CARCINOGEN IN CIGARETTE SMOKE ASSOCIATED WITH INCREASED CANCER RISK. WHILE BAP-INDUCED CARCINOGENESIS IS TRADITIONALLY ATTRIBUTED TO POINT MUTATIONS FROM ERRONEOUS BYPASS OF BENZO[A]PYRENE DIOL-EPOXIDE (BPDE)-DNA ADDUCTS DURING REPLICATION, OUR RECENT FINDINGS REVEAL AN ADDITIONAL PATHWAY: BPDE EXPOSURE GENERATES SINGLE- STRANDED DNA (SSDNA) GAPS DURING REPLICATION, WITH DNA SYNTHESIS RESTARTING DOWNSTREAM THROUGH PRIMPOL ACTIVITY. CRITICALLY, WE DISCOVERED THAT UNREPAIRED SSDNA GAPS TRANSFORM INTO DOUBLE-STRANDED DNA BREAKS (DSBS) WHEN THE PARENTAL STRAND IS CLEAVED BY MRE11 ENDONUCLEASE-REPRESENTING A PREVIOUSLY UNANTICIPATED FORM OF DNA DAMAGE INDUCED BY BAP. THIS PROJECT AIMS TO CHARACTERIZE HOW THESE BPDE-INDUCED DSBS MAY BE REPAIRED THROUGH NON-HOMOLOGOUS END-JOINING (NHEJ) PATHWAYS AND TO DETERMINE THEIR CONTRIBUTION TO GENOMIC INSTABILITY AND MUTAGENESIS. AS A GRADUATE STUDENT AT PENN STATE COLLEGE OF MEDICINE, THIS AWARD WOULD PROVIDE ME WITH THE MENTORSHIP, TRAINING IN ADVANCED DNA REPAIR TECHNIQUES, AND CAREER DEVELOPMENT OPPORTUNITIES NEEDED TO BUILD CRITICAL SKILLS FOR BECOMING AN INDEPENDENT SCIENTIST INVESTIGATING ENVIRONMENTAL CARCINOGENESIS MECHANISMS. IN THIS PROPOSAL I FOCUS ON ELUCIDATING HOW BPDE INDUCED DSBS CAN BE REPAIRED BY ERROR-FREE HOMOLOGOUS RECOMBINATION (HR) OR BY ERROR-PRONE NON-HOMOLOGOUS END JOINING (NHEJ). PRELIMINARY PROXIMITY LIGATION ASSAYS REVEALED KU80, WHICH INITIATES NHEJ, CO-LOCALIZES WITH DNA-BPDE ADDUCTS. I HYPOTHESIZE THAT BPDE-MEDIATED DSBS TRIGGER NHEJ REPAIR, CAUSING CHROMOSOMAL TRANSLOCATIONS AND GENOMIC INSTABILITY-A NOVEL MECHANISM OF BAP-INDUCED GENOMIC INSTABILITY. BENZO[A]PYRENE EXPOSURE IS A WELL-ESTABLISHED CANCER RISK, YET OUR PREVIOUS STUDIES INDICATE INCOMPLETE UNDERSTANDING OF MECHANISMS CONVERTING BPDE ADDUCTS INTO GENOMIC INSTABILITY. PROCESSING BPDE LESIONS INTO DSBS AND THEIR REPAIR VIA MUTAGENIC NHEJ MAY REPRESENT A CRITICAL PATHWAY IN BAP CARCINOGENESIS, POTENTIALLY REVEALING NEW TARGETS FOR CANCER PREVENTION. TO ADDRESS THIS HYPOTHESIS, I WILL EXPLORE TWO AIMS: 1) TO UNDERSTAND THE MECHANISM OF NHEJ ENGAGEMENT AT BPDE ADDUCTS; 2) TO UNDERSTAND THE IMPACT OF BPDE-INDUCED DSB REPAIR BY NHEJ. COMPLETING THESE AIMS WITH THIS AWARD WOULD EXPAND MY TECHNICAL EXPERTISE WHILE POTENTIALLY UNCOVERING A NOVEL MECHANISM OF GENOME INSTABILITY THROUGH NHEJ-MEDIATED REPAIR OF BPDE-INDUCED DOUBLE-STRANDED BREAKS.