A great deal of controversy surrounds the association between MSI (microsatellite 1 instability) and the response to 5-FU (5-fluorouracil) chemotherapy in patients with CRC (colorectal cancer). Nevertheless, it is widely accepted that MSI cancer patients have better survival and less metastasis compared to MSS (microsatellite stability) CRC patients, but whether MSI or MSS patients benefit differently from 5-FU therapy remains unclear. The MLH1 (mutL 5 homolog-1) gene defect tends to contribute to MSI in a major way, and it is one of the major causes of this condition. Many anticancer drugs are known to induce autophagy, including 5-FU however, it is currently unknown how MLH1 contributes to the autophagy-induced cellular response to anticancer drugs. We hypothesized that MLH1-proficient cancer cells could stimulate the autophagy of nuclear material (nucleophagy) as a defense mechanism to resist the cytotoxic effect of chemotherapy. In this study, we aimed to decipher the molecular link between the mismatch repair pathway protein MLH1 and the autophagy protein, microtubule-associated protein light chain 3 (LC3), which facilitated nucleophagy induction, and its potential role in determining sensitivity to 5-FU-induced cell death. To assess the role that MLH1 plays in DNA damage-induced nucleophagy in CRC cells, a panel of MLH1-deficient and MLH1-proficient CRC cell lines were used. In addition to the parental HCT116 cell line (MLH1-/-), we also used the HCT116 MLH1+/- cell line, which carried a single allele of the MLH1 gene introduced by CRISPR/Cas9 gene editing. We provided evidence that MLH1-proficient CRC cells were more 18 resistant to 5-FU-induced DNA damage in comparison to MLH1-deficient CRC cells. Moreover, in MLH1-proficient cells, 5-FU induced DNA damage, causing upregulation of the autophagy protein LC3. The immunofluorescence assay revealed MLH1 and LC3 colocalize in CRC cells, which was further supported by our immunoprecipitation assay, which showed that 5-FU treatment promotes MLH1 and LC3 molecu