Chromatin Remodeling Histone Chaperone Fact Complex Modulates Ap Site Damage Repair In Chromatin And Sensitizes Cancer Cells To Chemotherapeutic Drugs

Recognition and repair of DNA lesions in the genome are critical for maintaining genomic stability and reducing the generation of mutations that lead to cancer development. The most frequently formed DNA lesion in the genome is the apurinic/apyrimidinic (AP) site which is mutagenic and blocks transcription and replication. The primary enzyme to repair AP sites in mammalian cells is the AP endonuclease (APE1), which often overexpresses in diverse cancer types and its overexpression is associated with patients’ resistance to chemotherapeutic drugs. The repair of AP sites by APE1 through the Base Excision Repair (BER) pathway is extensively investigated in vitro. However, how AP site is recognized by APE1 in the context of highly complex nucleosome structure in chromatin is unknown. Because DNA is packaged tightly in nucleosome, the ability of repair proteins to access sites of DNA damage and facilitates repair of the damage requires chromatin remodeling activities. Here, we show that APE1 interacts with chromatin remodeling histone chaperone complex, FACT (facilitates of chromatin transcription) via its N-terminal domain in cells. By immunoprecipitation of endogenous APE1 from chromatin fraction and separation of protein bands in SDS-PAGE followed by MALDI-TOF-TOF analysis, we have identified both subunits (SPT16 & SSRP1) of FACT as the prominent APE1 interacting partners. Subsequently, we confirmed the interaction of APE1 with FACT complex by Co-IP and immunofluorescence analysis and found that both SPT16 and SSRP1 interact with APE1 in the nucleus and in chromatin. Interestingly, we found rapid eviction of histones with concomitant degradation of FACT complex upon induction of DNA damages. Downregulation of FACT complex abrogates the nucleosome eviction, the recruitment of repair protein APE1 in chromatin and the repair of AP sites, demonstrating the functional importance of nucleosomes disruption in BER pathway and identifying chromatin remodeling protein required for the process. Notably, knock-down of FACT showed increased sensitivity of cancer cells to many chemotherapeutic drugs. We also found that FACT down-regulated cells have much higher accumulation of AP site damages in the genome compared to control cells. Our study revealed a key role of nucleosome remodeling complex FACT in DNA damage repair in BER pathway. This study also suggests that histone chaperone complex could be a potential target for enhancing sensitivity of tumor cells to chemotherapy. Citation Format: Heyu Song, Shrabasti Roychoudhury, Dan Feng, Kishor Bhakat. Chromatin remodeling histone chaperone FACT complex modulates AP site damage repair in chromatin and sensitizes cancer cells to chemotherapeutic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1414. doi:10.1158/1538-7445.AM2017-1414