The long-term goal of this project is to determine how defects in flap endonuclease 1 (FEN1)-mediated Okazaki fragment maturation (OFM) cause genomic instability and contribute to human cancer initiation, progression, and drug resistance. In the lagging strand DNA synthesis process, proofreading deficient primase and polymerase ? (Pol ?) synthesize the RNA primers and the DNA fragment connected to the primers (?- segment). OFM is a fundamental mechanism for faithful DNA replication, which can be divided into two steps: RNA primer removal (RPR) and ?-segment error editing (AEE). Recent data indicate that the ?-segment accounts for approximately 1.5% of the genome. Therefore, defective OFM can be a significant source of DNA mutations. Our recent published work indicates that AEE depends on FEN1?s exonuclease (EXO) activity, whereas RPR requires its structure-specific endonuclease (FEN) activity. In addition, sequential post- translational modifications (PTMs), which mediate FEN1?s interactions with other DNA replication machinery components, such as PCNA, WRN, and MutS?, are crucial for highly organized OFM. However, many questions remain unanswered. How is the FEN1-mediated OFM process sequentially coordinated among the major enzyme actions and with downstream histone deposition? How is the function of OFM complexes influenced by cellular stresses, such as chemo- and radio- therapeutic stresses? Consequently, how does the dysfunctional OFM complex promote therapeutic resistance? Based on our exciting preliminary data, we hypothesize that programmed PTMs of FEN1, dynamic interaction between the OFM machinery and histone deposition, and HSP70-mediated OFM complex coordination are key regulatory mechanisms for efficient and accurate FEN1-mediated OFM. Alterations in these regulatory mechanisms may impair OFM and dramatically increase the mutation frequency, cancer predisposition, and development of drug resistance. We will test the hypothesis with the following specific aims: 1) To determine the role of arginine demethylase (JMJD1B)- mediated FEN1 demethylation in OFM. 2) To determine if the dynamic balance between FEN1-mediated OFM and histone deposition, assembly, and positioning ensures accurate replication of genetic and epigenetic information. 3) To determine if HSP70 is critical for the correct assembly of functional OFM complexes to suppress DNA mutations and chemotherapeutic resistance. The current renewal application is based on successful completion of all the proposed work for the last cycle of funding with 13 peer-reviewed publications, including publications in high-impact journals, establishment of state-of-the-art experimental systems and exciting preliminary data that support the paradigm-shifting hypotheses. We are in a unique position to address these questions because of our initial observations and the experimental systems established in my laboratory.

Public Health Relevance

The overall goal of this project is to determine how the flap endonuclease FEN1 directs Okazaki fragment maturation (OFM) during lagging strand genomic DNA replication, while restricting the formation of potentially carcinogenic mutations. The proposed work will test the role of a novel arginine demethylase in FEN1- mediated OFM, establish the inter-regulation between OFM and histone deposition, and evaluate the role of HSP70 in coordinating OFM complex stability and suppressing the development of chemotherapy-induced secondary mutations. The information obtained from the current studies will significantly enhance our understanding of cancer etiology, and may produce molecular targets for novel therapeutic regimens and diagnostic or prognostic markers.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Radiation Therapeutics and Biology Study Section (RTB)
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Okano, Paul
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Beckman Research Institute/City of Hope
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Li, Min; Xu, Xiaohua; Chang, Chou-Wei et al. (2018) SUMO2 conjugation of PCNA facilitates chromatin remodeling to resolve transcription-replication conflicts. Nat Commun 9:2706
Qian, Shu-Wen; Wu, Meng-Yuan; Wang, Yi-Na et al. (2018) BMP4 facilitates beige fat biogenesis via regulating adipose tissue macrophages. J Mol Cell Biol :
Cao, Xiang; Zhou, Yi; Sun, Hongfang et al. (2018) EGFR-TKI-induced HSP70 degradation and BER suppression facilitate the occurrence of the EGFR T790?M resistant mutation in lung cancer cells. Cancer Lett 424:84-96
Li, Sihui; Ali, Shafat; Duan, Xiaotao et al. (2018) JMJD1B Demethylates H4R3me2s and H3K9me2 to Facilitate Gene Expression for Development of Hematopoietic Stem and Progenitor Cells. Cell Rep 23:389-403
Sun, H; He, L; Wu, H et al. (2017) The FEN1 L209P mutation interferes with long-patch base excision repair and induces cellular transformation. Oncogene 36:194-207
Zhou, Lina; Dai, Huifang; Wu, Jian et al. (2017) Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development. FASEB J 31:132-147
Zhang, Yijuan; Wen, Chunhong; Liu, Songbai et al. (2016) Shade avoidance 6 encodes an Arabidopsis flap endonuclease required for maintenance of genome integrity and development. Nucleic Acids Res 44:1271-84
Zhou, Ting; Pan, Feiyan; Cao, Yan et al. (2016) R152C DNA Pol ? mutation impairs base excision repair and induces cellular transformation. Oncotarget 7:6902-15
Ali, Shafat; Zheng, Li; Shen, Binghui (2015) FEN1-mediated ?-segment error editing during Okazaki fragment maturation. Cell Cycle 14:1767
Liu, Songbai; Lu, Guojun; Ali, Shafat et al. (2015) Okazaki fragment maturation involves ?-segment error editing by the mammalian FEN1/MutS? functional complex. EMBO J 34:1829-43

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