Exploring the effects of RNase H2 regulation and Protein Lysine Acetylation on genomic stability

Maintenance of genomic integrity during DNA replication requires precise coordination of multiple enzymatic processes. The antiparallel nature of the duplex DNA necessitates asymmetric replication, where the leading strand is synthesized continuously while the lagging strand is synthesized discontinuously as Okazaki fragments. In human cells, approximately 50 million Okazaki fragments are generated per replication cycle and must be matured and seamlessly joined to produce an intact lagging strand. Defects in this process contribute to genomic instability and have been implicated in cancer development. The Okazaki fragment maturation (OFM) process involves a coordinated action of several key enzymes: DNA Polymerase δ (Pol δ) synthesizes and displaces the preceding fragment creating a flap, which is then processed by flap endonuclease 1 (FEN1) and the remaining nick is sealed by DNA LigI. Additionally, despite their high fidelity, replicative polymerases can occasionally incorporate ribonucleotides during synthesis, which is processed by Ribonuclease H2 (RNase H2) in the ribonucleotide excision repair (RER) pathway. Central to both pathways is proliferating cell nuclear antigen (PCNA), a sliding clamp that mediates these activities through protein-protein interactions via PCNA-interaction peptide (PIP) domains. The fidelity and efficiency of lagging strand synthesis therefore depend critically on the sequential handoff of DNA substrate between these coordinated enzymes. This study investigates two regulatory mechanisms that modulate protein function and influence pathway efficiency and overall genome stability. First, we examined RNase H2’s role as a potential coordinator linking OFM and RER pathways. Our results reveal bidirectional stimulatory interactions between RNase H2 and key downstream enzymes - Pol δ, FEN1, and DNA LigI, suggesting a coordinated regulatory network. Second, we investigated how lysine acetylation, a dynamic post-translational modification, affects the enzymatic activity of RNase H2, Pol δ, and RPA. In vitro acetylation studies demonstrate enhanced enzymatic activity for all three proteins, indicating that this modification may serve as a governing mechanism controlling OFM efficiency. Collectively, our findings suggest that these protein interaction networks, and regulatory mechanisms offer potential therapeutic targets for cancer and other diseases associated with genomic instability.