Project Grant F31CA318260

MECHANISMS AND DNA TARGETING OF LINE-1 ORF2P ENDONUCLEASE - PROJECT SUMMARY/ABSTRACT LONG INTERSPERSED NUCLEAR ELEMENT-1 (L1) IS THE ONLY ACTIVE AND AUTONOMOUS RETROTRANSPOSON IN THE HUMAN GENOME, GENERATING NEW INSERTIONS THROUGH A "COPY-AND-PASTE" MECHANISM EMPLOYING TARGET-PRIMED REVERSE TRANSCRIPTION (TPRT). DEREPRESSION OF L1 TRANSCRIPTION, AND RESULTANT INCREASE IN L1 ACTIVITY, IS LINKED TO A WIDE RANGE OF HUMAN DISORDERS, INCLUDING CANCER, NEURODEGENERATION, AUTOIMMUNE DISEASE, AND AGE-ASSOCIATED PATHOLOGIES, UNDERSCORING THE NEED TO DEFINE THE MECHANISMS BY WHICH L1 PROTEINS MANIPULATE DNA. A CRITICAL BUT POORLY UNDERSTOOD STEP IN THIS PROCESS IS THE INITIAL DNA NICK GENERATED BY THE ENDONUCLEASE (EN) DOMAIN OF ORF2P, WHICH CREATES THE 3 HYDROXYL REQUIRED TO PRIME REVERSE TRANSCRIPTION. RECENT FINDINGS INDICATE THAT EN IS FAR MORE PERMISSIVE IN ITS SUBSTRATE SELECTION THAN PREVIOUSLY RECOGNIZED AND THAT MOST TPRT ATTEMPTS FAIL, GENERATING ABORTIVE DNA INTERMEDIATES RATHER THAN PRODUCTIVE INSERTIONS. HOWEVER, THE STRUCTURAL AND BIOCHEMICAL PRINCIPLES GOVERNING HOW EN ENGAGES GENOMIC DNA AND PROCESSES THE RESULTING CLEAVAGE PRODUCTS REMAIN LARGELY UNKNOWN. THE LONG-TERM GOAL OF THIS PROJECT IS TO DEFINE THE MECHANISTIC BASIS BY WHICH EN INITIATES DNA DAMAGE AND THEREFORE SHAPES L1-CONTRIBUTION TO DISEASE. AIM 1 WILL DEFINE THE STRUCTURAL DETERMINANTS OF DNA BINDING AND CLEAVAGE BY EN, USING BOTH IN VITRO AND CELL-BASED ASSAYS. ALTHOUGH EN IS OFTEN DESCRIBED AS TARGETING A NARROW CONSENSUS SEQUENCE, BIOCHEMICAL DATA INDICATE THAT DNA STRUCTURAL FEATURES STRONGLY INFLUENCE CLEAVAGE EFFICIENCY. THIS WORK WILL IDENTIFY THE DNA CONFORMATIONAL PROPERTIES THAT PROMOTE EN ACTIVITY AND DETERMINE HOW THESE FEATURES IMPACT ACTIVE-SITE CONFIGURATION AND SUBSTRATE ENGAGEMENT. BY EXPRESSING EN MUTANTS IN CELLS AND ASSESSING DNA DAMAGE AND GENOMIC BINDING PROFILES, WE WILL ESTABLISH HOW SPECIFIC FUNCTIONAL CHANGES IN EN ALTER ITS BIOLOGICAL CONSEQUENCES. AIM 2 WILL DETERMINE THE KINETIC AND STRUCTURAL BASIS OF EN INTERACTIONS WITH ITS CLEAVAGE PRODUCTS AND EVALUATE HOW PRODUCT RELEASE RATES IMPACT PRIMING OF REVERSE TRANSCRIPTION IN THE CONTEXT OF FULL- LENGTH ORF2P. BECAUSE THE 3-HYDROXYL GENERATED BY EN MUST TRANSFER FROM THE EN CATALYTIC SITE TO THE RT DOMAIN, THE TIMING AND DYNAMICS OF PRODUCT RELEASE ARE LIKELY TO DICTATE WHETHER TPRT PROCEEDS EFFICIENTLY OR TERMINATES IN ABORTIVE DNA INTERMEDIATES. THIS FELLOWSHIP WILL BE CARRIED OUT AT BROWN UNIVERSITY, WHICH OFFERS OUTSTANDING STRUCTURAL BIOLOGY, BIOCHEMISTRY, AND IMAGING CAPABILITIES. GUIDED BY DR. MARTIN TAYLOR, CO-MENTOR DR. GERWALD JOGL, AND A HIGHLY ENGAGED TEAM OF COLLABORATORS AND ADVISORS COMPOSING THE CANDIDATE'S THESIS COMMITTEE, THE TRAINING PLAN COMBINES ADVANCED STRUCTURAL METHODS, CELL-BASED ASSAYS, AND QUANTITATIVE BIOPHYSICS WITH TARGETED PROFESSIONAL DEVELOPMENT. WITHIN THE COLLABORATIVE COMMUNITY OF BROWN'S CENTER FOR THE BIOLOGY OF AGING, THE APPLICANT WILL HAVE THE SUPPORT AND RESOURCES NEEDED TO BUILD A STRONG FOUNDATION IN STRUCTURAL BIOLOGY, GENOME STABILITY, AND RETROTRANSPOSON BIOLOGY, AND TO DEVELOP INTO AN INDEPENDENT INVESTIGATOR.