Friday, April 18, 2025
4/18/2025
Roles and regulations of APE1 in ATR signaling - Project Summary/Abstract Genome of all cells are constantly exposed to endogenous insults such as reactive oxygen species and environmental toxins, leading to different types of DNA damage such as Apurinic/apyrimidinic (AP) sites, single-strand breaks (SSBs), and single-strand DNA (ssDNA) gaps. Although a good progress has been made on how a global ATR-mediated checkpoint signaling is triggered by DNA damage, it remains elusive how the ATR signaling is activated by ssDNA gaps derived from DNA damage and replication stress, and in the nucleoli where the RNA polymerase I-mediated rRNA transcription and ribosome assembly take place. Whereas multifunctional protein APE1 has been involved in base excision repair, redox regulation, and RNA processing, it remains unclear how APE1 plays critical functions in nucleolar ATR signaling and how APE1 recognizes and senses ssDNA gaps for the activation of ATR signaling. Our substantial preliminary data suggest that APE1 assembles into distinct liquid-liquid phase separation (LLPS) in vitro and in the nucleoli of cultured cancer cells but not normal cells to promote the ATR-Chk1 signaling, leading to compromised ribosomal RNA transcription and reduced cell viability. Furthermore, APE1 plays a direct role in the recognition and association of ssDNA gaps to recruit the ATR/ATRIP complex in in vitro reconstitution system and to promote the ATR checkpoint signaling in Xenopus egg extracts. Our central hypothesis in this project is that APE1 forms LLPS in vitro and in the nucleoli to promote the ATR checkpoint signaling and that APE1 directly recognizes ssDNA gaps for the recruitment and activation of ATR/ATRIP complex. We will elucidate the distinct regulatory mechanisms of APE1 in ATR signaling via two Specific Aims: (1) to determine the mechanisms of how APE1 forms liquid-liquid phase separation in vitro and in nucleoli to sense pH in cancer cells but not normal cells and promotes the nucleolar ATR-Chk1 checkpoint signaling to regulate rDNA transcription and sensitivity to chemotherapy drugs, and (2) to determine how APE1 directly associates with ssDNA gaps to promote the global ATR-Chk1 signaling in oxidative stress and DNA replication stress in Xenopus egg extracts and mammalian cells. We will test our major hypothesis regarding distinct mechanisms of APE1 in ATR DDR signaling using cutting-edge molecular biology, cell biology, and biochemical approaches in in vitro reconstitution systems with purified proteins, Xenopus egg extracts, and cultured mammalian cells. Anticipated results from this project will make a paradigm shift on how APE1 regulates ATR checkpoint signaling in the nucleoli and how APE1 recognizes and senses ssDNA gaps for the activation of the global ATR-Chk1 signaling to maintain genome stability. This work will provide novel insights into new strategies for cancer therapy such as modulating the distinct regulatory mechanisms of APE1 in ATR signaling in cancer cells.
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