Abstract The proposed R50 award will support my critical contributions to three cohesive research programs conducted in the laboratory of Dr. Binghui Shen at the Beckman Research Institute of City of Hope: NCI R01 CA073764 (1997-2022), NCI R01 CA085344 (1999-2024), and NCI R01 CA233664 (2019-2024). Various DNA intermediates are formed during DNA metabolic processes, and these structures must be properly and efficiently processed in a timely manner to avoid severe genomic instability and cancer development. This process requires structure-specific nucleases to specifically recognize and cleave certain forms of DNA structures for further processing and eventual DNA ligation into an intact DNA duplex. Over the last 20 years, I have supported the Shen laboratory in investigating the structure, function, and regulation of structure-specific nucleases typified by flap endonuclease 1 (FEN1) and DNA2 nuclease/helicase, as well as how their functional deficiency causes genome instabilities and cancer development. More recently, our research has expanded to characterizing the role of exonuclease 5 (EXO5) in resecting androgen receptor-induced double-strand breaks in prostate cancer. As the senior research scientist in the Shen laboratory, I have leveraged my expertise in biochemical and cellular assays of DNA replication and repair, mouse genetics, and cutting-edge next-generation sequencing technologies to continuously contribute to breakthrough findings that advance Dr. Shen’s research programs. Since I joined Dr. Shen’s group in 2001, I have co-authored 50 peer-reviewed publications, including 17 peer- reviewed articles resulting from my R50-sponsored research in the previous funding period (2016-2021). Currently, I am supporting the CA073764 program by defining post-translational modifications (PTMs) of FEN1 and the dynamic interaction between Okazaki fragment maturation (OFM) and histone deposition machinery as key regulatory mechanisms for efficient and accurate FEN1-mediated OFM. The current goal of the CA085344 program is to define the roles of human DNA2 in counteracting replication stress. In support of this, I will lead a team to determine how human DNA2 complexes process G4 and other non-B-form DNA structures to facilitate DNA replication at telomeres and centromeres, using DNA2 mutant mouse models. I am also directing a portion of the proposed research in the CA233664 program, which aims to define the etiological role of homology- directed repair (HDR) gene mutations in prostate cancer. I created knockout mice for the EXO5 gene, which we recently discovered to play a crucial role in HDR in prostate cells. I will lead the effort to define the causal role of EXO5 in prostate cancer and reveal the underlying molecular events. These studies will leverage my knowledge and experience accumulated during my previous research in FEN1 and DNA2. As an NCI-sponsored research specialist, I have and will continue to maximize my contribution to science by le...