Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

Double-strand breaks (DSBs), which are among the most dangerous DNA lesions, are estimated to occur at a rate of ten per cell per day in primary human or mouse fibroblasts. These naturally occurring DSBs are generated upon collapse of stalled DNA replication forks, replication across nicks, reactive oxygen species of endogenous origin or the untimely action of DNA endonucleases. DSBs are mainly repaired by non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the primary pathway and is used throughout the cell cycle, while HR is active in S/G2 phases where sister chromatids are available as repair templates. The regulation of DSB repair pathway choice is of great importance to our cells since errors in the choice of DSB repair pathway can create gene rearrangements and faulty DSB repair often generates aberrant chromosomal structures that kill cells. NHEJ is active only on minimally processed DNA ends; it is inhibited by DNA end resection, the process by which 5’-3’ nucleolytic degradation generates single-stranded DNA (ssDNA). The 3'-ssDNA overhangs are immediately coated by the Replication Protein A (RPA) protein complexes (composed of subunits of ~70, 32, and 14 kDa, referred as to RPA70, RPA32, and RPA14, respectively) for protection. The RPA-coated ssDNA is an essential intermediate of all HR sub-pathways. CtBP-interacting protein (CtIP) is a key DNA endonuclease controlling DNA end resection. CtIP is targeted by multiple posttranslational modifications (PTMs) including phosphorylation, ubiquityation, acetylation and other less known modifications.
Protein SUMOylation has recently emerged as a PTM critical for DSB repair. A group of proteins involved in HR, including Breast Cancer 1 (BRCA1), Mediator Of DNA Damage Checkpoint 1 (MDC1), RPA70, and Bloom syndrome protein (BLM), is directly modified by SUMO. Although it is evident that protein SUMOylation plays an important role in DNA repair, whether SUMOylation is directly involved in DNA end resection is still unclear. In the present study, we aim to determine how SUMOylation/de- SUMOylation regulates CtIP's function in DNA end resection.
In this application, we have found that CtIP is SUMOylated at specific lysine residue in response to DNA damage. In the first objective , we will characterize the function(s) of this sumoylation site in HR. In the second objective , we will determine the possible interplay between CtIP SUMOylation and other existing CtIP PTMs. We also identified the main de-sumoylating enzyme that regulates CtIP SUMOylation. In the third objective , we will determine the impact of CtIP de-SUMOylation on its function in HR. If successful, our studies will greatly advance our understanding of the mechanism of DNA end resection, the role of CtIP SUMOylation in DNA repair, and the interplay between SUMOylation and other existing PTMs of CtIP.