Abstract 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.