UHRF1-DNMT1 Cooperation in Cytosine Base Flipping and Maintenance Methylation: Mechanistic and Oncogenic Insights from HeLa Cells

Aberrant DNA methylation is a defining characteristic of cancer epigenetics. In cervical cancer, the dysregulation of methylation maintenance leads to the abnormal suppression of tumor-suppressor genes. DNA methyltransferase 1 (DNMT1), Ubiquitin-like containing PHD, and RING Finger Domains 1 (UHRF1) work together to maintain methylation during DNA replication. Using its SRA domain, UHRF1 identifies hemi-methylated CpG sites, flips the unmethylated cytosine base, and enlists DNMT1 to remethylate the emerging strand. High-resolution Leica SP8 confocal microscopy was employed to visualize this interaction using HeLa cells via EGFP-DNMT1 and mCherry-UHRF1 constructs with EdU incorporation. Coexpression enhanced EdU- positive nuclei and colocalisation (R = 0.68 ± 0.07), whereas the UHRF1 R491A mutant impaired DNMT1 recruitment (R = 0.33 ± 0.05). These results demonstrate the pathogenic amplification of cytosine base flipping in cervical cancer and validate it as a crucial phase in replication-coupled methylation. Focusing on this process may provide a new therapeutic approach for epigenetic reprogramming. Keywords: DNMT1, UHRF1, cytosine base flipping, confocal microscopy, cervical carcinoma