INVESTIGATING THE LINK BETWEEN HOMOLOGOUS RECOMBINATION PROTEIN BRCA2 AND DNA METHYLATION

Cancer cell genomes display numerous mutation patterns that subvert genome stability. DNA repair and damage response mechanisms are responsible for maintaining stability in normal cells. One such repair mechanism involves homologous recombination (HR) proteins BRCA2 which protects DNA replication intermediates from nuclease-mediated degradation. Emerging evidence demonstrates that HR proteins like BRCA2 co-evolved with the cytosine methylation machinery, suggesting a functional link between the two processes. Cytosine methylation is an epigenetic modification occurring across many lifeforms, primarily associated with the repression of gene expression. However, its influence on DNA replication and repair remains largely unknown. Here, we investigate the presence and mechanism of a functional link between the two processes. To overcome the lack of single-molecule tools capable of monitoring methylation dynamics, we developed and validated a hemimethylation-discriminating DNA fiber assay using the restriction endonuclease NhoI. By using this assay, we demonstrated that BRCA2 expression did not affect the function of DNMT1, the protein responsible for post replicative DNA methylation maintenance. We subsequently investigated whether DNMT1 influenced DNA replication and repair pathways. To this end, we employed an auxin-induced degron (AID) system for DNMT1 for its reversible degradation. Through the DNA fiber assay on this cell line, we demonstrated that the loss of cytosine methylation results in faster replication fork progression, revealing methylation to act as a rheostat for DNA replication. We then investigated if this impacted replication fork stability under replication stress conditions. Specifically, we investigated whether this impacted the nucleolytic degradation of the nascent strand to hydroxyurea, which usually occurs in HR-defective cells. Using DNA fiber assay, we demonstrated that the HU-induced nascent strand degradation conferred by the loss of BRCA2 is completely reliant on cytosine methylation. Upon further investigation, we observed that the cytosine methylation turnover proteins like SMUG1 partially contribute to the degradation of the nascent strands, while nucleotide excision repair proteins like DDB1 does not contribute. Taken together, our model suggests that the abasic site originating from cytosine methylation turnover partially results in the vulnerability of the nascent strand for nuclease-mediated degradation. Targeting the histone methylation pathways, particularly H3K9Me3 revealed a strong counter-effect to the protection offered by the loss of DNA methylation, suggesting that chromatin state dictates fork vulnerability to nucleases. Our findings outline a model where cytosine methylation promotes genome stability by regulating fork speed and coordinating the fork reversal process for replication stress tolerance through histone modifications. Conversely, our findings offer an explanation as to why advancing cancers undergo global hypomethylation, thereby promoting genome instability. Lastly, this study also opens an avenue for treating hypomethylated cancers by targeted enhancement of H3K9Me3 formation.