In this study, we addressed the question by using human colon cancer HCT116-BKO cells in which two alleles of DNMT3b disrupted by homologous recombination and ICF cells that carry loss-of-function mutations in DNMT3b. Our analysis demonstrated that R-loops contribute to prominent DNA damage signals observed in both DNMT3b knockout in HCT116 (BKO) and the loss-of-function mutation of DNMT3b in ICF lymphocytes. Here, DNMT3B is linked to colonic neoplasm.