DNA Base Excision Repair Modulates DNA Repeat Instability and Non-B Form DNA Structures

Abstract

The human genome is constantly attacked by endogenous and exogenous sources of DNA damage that generates DNA base lesions and strand breaks leading to genome instability, cell death, and diseases. To combat these adverse effects, cells have evolved a robust DNA repair mechanism called “the DNA base excision repair (BER),” which efficiently removes DNA lesions maintaining genome stability. However, its underlying molecular mechanisms remain to be elucidated. In my dissertation research, I explored the molecular mechanism by which BER modulates trinucleotide repeats (TNR) by processing non-B form structures such as hairpins and R-loops through the coordination among BER enzymes and cofactors and the proteins from other DNA repair pathways. For the first time, we discovered that Fanconi anemia-associated nuclease 1 (FAN1) coordinated with a key BER enzyme, flap endonuclease 1 (FEN1), to prevent repeat expansion by promoting CAG repeat deletion during BER of oxidative DNA damage. We further demonstrated that the BER cofactor, PCNA/ubPCNA, coordinated with FAN1 to attenuate CAG repeat expansion during BER in a CAG repeat hairpin. Exploring the mechanisms by which BER regulates TNR instability by processing bulky non-B form structures, R-loops. We found that an abasic lesion on the non-template strand of a CAG repeat R-loop was incised by AP endonuclease 1 (APE1), converting the R-loop into a double-flap intermediate containing an RNA:DNA hybrid. This inhibited DNA polymerase β (pol β) DNA synthesis and stimulated FEN1 cleavage of the repeats leading to repeat deletion. We showed that FEN1 partially cleaved the RNA strand on a CAG repeat R-loop to facilitate pol β skip-over of a hairpin leading to repeat deletion. We further identified a new role of FEN1 of resolving R-loops through the BER pathway. We found that FEN1 efficiently cleaved RNA during the processing of DNA lagging strand and R-loops and demonstrated that FEN1 also used its flap tracking to track down to the DNA region to make a cleavage removing the RNA leading to the lagging strand maturation and R-loop resolution via BER. Our study provides new insights into the molecular mechanism by which BER maintains genome stability and integrity.