Project Grant F31AG092149

DECIPHERING THE FUNCTION OF COHESIN AND CTCF IN R-LOOP-MEDIATED DNA REPAIR AT TELOMERES - R-LOOPS (RLS) ARE TRIPLE-STRANDED NUCLEIC ACID STRUCTURES CONSISTING OF AN RNA-DNA HYBRID AND A DISPLACED SINGLE-STRANDED DNA LOOP. WHILE UNREGULATED RL ACCUMULATION CAUSES GENOMIC STRESS, DNA DAMAGE-INDUCED RLS CAN ALSO REGULATE THE DNA DAMAGE RESPONSE (DDR). WHILE EXCESSIVE TELOMERIC R-LOOP (TRL) ACCUMULATION IS LINKED TO TELOMERE SHORTENING, AND THUS, AGING, MECHANISTIC UNDERSTANDING OF THE FUNCTION OF TRLS IN THE DDR AT TELOMERES IS LIMITED. RECENT FINDINGS FROM OUR LAB AND MY CO-SPONSOR, DR. LI LAN (DUKE UNIVERSITY), IMPLICATE THE COHESIN-NIPBL COMPLEX AND ITS COFACTOR, CCCTC-BINDING FACTOR (CTCF), IN TRL- MEDIATED REPAIR AT TELOMERES. OUR PRELIMINARY ATOMIC FORCE MICROSCOPY (AFM) DATA ESTABLISHES COHESIN AND CTCF AS AVID R-LOOP BINDING PROTEINS, AND WE SHOW COHESIN'S SA2 SUBUNIT BINDS TO TRLS. USING A SYSTEM THAT INDUCES SITE-SPECIFIC R-LOOPS IN A DAMAGE-DEPENDENT FASHION IN HUMAN CELLS (KILLERRED SYSTEM), DR. LAN'S LAB SHOWS THAT COHESIN RECRUITS THE RAD51 REPAIR PROTEIN TO GENOMIC DAMAGE SITES THROUGH R-LOOPS. ADDITIONALLY, THEY SHOW COHESIN'S SA1 AND SA2 SUBUNITS LOCALIZE TO TRLS INDUCED SPECIFICALLY AT TELOMERES. OUR PRELIMINARY DATA RAISES THE POSSIBILITY THAT COHESIN AND CTCF FACILITATE THE TELOMERE DDR BY RECOGNIZING DNA DAMAGE- INDUCED TRLS AND RECRUITING REPAIR PROTEINS TO DAMAGED TELOMERE SITES. IN THIS PROPOSED WORK, I WILL TEST THIS HYPOTHESIS BY LEVERAGING A UNIQUE COMBINATION OF IN VITRO AFM IMAGING AND FLUORESCENCE CELLULAR IMAGING WITH THE KILLERRED SYSTEM. IN AIM 1, TAKING ADVANTAGE OF THE NANOMETER RESOLUTION OF AFM, I WILL CHARACTERIZE THE MOLECULAR MECHANISM BY WHICH COHESIN AND CTCF INTERACT WITH TRLS BY IDENTIFYING THE COHESIN SUBUNITS INVOLVED IN TRL BINDING AND THE ROLE OF CTCF IN TRL LOCALIZATION BY COHESIN. IN AIM 2, I WILL USE A TELOMERE- SPECIFIC KILLERRED SYSTEM THAT INDUCES LOCALIZED DNA DAMAGE AND TRLS AT TELOMERES TO DETERMINE THE FUNCTION OF COHESIN AND CTCF IN TRL-MEDIATED DNA REPAIR AT TELOMERES IN HUMAN CELLS. I WILL DETERMINE IF DAMAGE- INDUCED TRLS ENHANCE COHESIN AND CTCF LOCALIZATION TO DAMAGED TELOMERES. FURTHERMORE, I WILL DETERMINE IF COHESIN AND CTCF RECRUIT REPAIR PROTEINS TO DAMAGED TELOMERES VIA TRLS USING MULTICOLOR FLUORESCENCE IMAGING. I PREDICT THAT DNA DAMAGE-INDUCED TRLS WILL BE RESOLVED UPON COMPLETION OF DNA REPAIR MEDIATED BY COHESIN AND CTCF, AND COMPROMISED RECRUITMENT OF COHESIN AND CTCF AT TRL WILL LEAD TO DNA REPAIR DEFECTS AND TRL ACCUMULATION. TO TEST THIS HYPOTHESIS, I WILL ASSESS IF THE REDUCTION IN COHESIN AND CTCF LEVELS WITH SIRNA PROMOTES TRL ACCUMULATION. LASTLY, PERSISTENT TELOMERE DAMAGE IS KNOWN TO ACCELERATE TELOMERE SHORTENING AND, WITH IT, CELLULAR AGING. I WILL ASSESS IF COHESIN AND CTCF MAINTAIN TELOMERE LENGTH IN A TRL- DEPENDENT FASHION BY MONITORING TELOMERE LENGTHS IN HUMAN CELLS WITH KNOCKDOWN OF COHESIN OR CTCF. THROUGH THE PROPOSED WORK, I WILL DECIPHER A PREVIOUSLY UNEXPLORED FUNCTION OF COHESIN AND CTCF IN TRL- MEDIATED DNA REPAIR AT TELOMERES, OPENING A NEW WINDOW INTO THE WORLD OF TELOMERE INSTABILITY AND AGING.