Thursday, November 21, 2024
11/21/2024
Project Summary The long-term goal of this project is to define mechanisms that regulate Okazaki fragment maturation (OFM) and determine how functional alterations in OFM enzymes and/or regulatory mechanisms cause genome instability and cancer. It is known that OFM processes are implemented via the scaffold protein PCNA, which mediates the sequential actions of Pol , flap endonuclease 1 (FEN1), and DNA ligase 1 (LIG1), in the DNA replication machinery. However, there are important knowledge gaps regarding the specific regulatory mechanisms that ensure the various OFM enzymes act sequentially to minimize mutagenesis and to control cell death. Unanswered questions include: How is Pol -mediated displacement DNA synthesis (SDDS) regulated by PARP1 to produce proper RNA-DNA 5' flap substrates for the nuclease FEN1? How are the sequential posttranslational modifications of FEN1 including arginine demethylation implemented to ensure its exit from the “work site” in late S phase? How are FEN1 and LIG1 properly exchanged onto the PCNA platform to ensure orderly OF access and suppression of an alternative, error-prone ligation process? In our preliminary studies, we have revealed mechanistic insights underlying OFM: i) PARP1 localizes to replication forks at early/middle but not late S phase, controlling SDDS by Pol. Its inhibition causes accumulation of 5' flaps in cells. ii) FEN1 arginine demethylation allows its dissociation from PCNA. JMJD1B is the arginine demethylase that mediates FEN1 arginine demethylation. JMJD1B localization to replication sites depends on histone chaperone FACT. iii) FEN1 and LIG1 sequentially co-localize with PCNA, allowing their orderly access to DNA substrates. iv) FEN1 S187A mutation or JMJD1B deletion similarly leads to PARP1 accumulation, histone PARylation, and LIG3 recruitment, similar to LIG1 knockdown cells. PARP1 inhibition is synthetically lethal with LIG1 knockdown. Based on these findings, we hypothesize that PARP1-regulated SDDS by Pol, FACT-mediated JMJD1B recruitment to demethylate FEN1 for its proper drop-off, and PCNA-coordinated FEN1 and LIG1 sequential access to OFs are crucial regulatory mechanisms for efficient and accurate OFM processes. Therefore, human cells with gene mutations and/or under stress conditions that disrupt these regulatory mechanisms will induce alternative, potentially faulty OFM processes for survival, leading to genome instabilities, cancer predisposition, and development of resistance to cancer therapy. To elucidate these regulatory mechanisms, we aim to i) determine the roles of PARP1 in controlling Pol -mediated SDDS to ensure proper formation of short 5' flaps for FEN1 cleavage; ii) determine how FACT-mediated arginine demethylase JMJD1B recruitment regulates FEN1 dynamics at replication forks; and iii) define the mechanism by which PCNA coordinates orderly access of FEN1 and LIG1 to OFs and suppresses an alternative, LIG3-mediated OF ligation process. gained will improve our understanding of cancer etiology and may inform treatment. The knowledge
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