DNA methylation is one of the major epigenetic modifications regulating biological processes such as normal development, chromosomal stability, and gene regulation. Aberrant hypermethylation of promoter CpG islands is an important mechanism for the inactivation of tumor suppressor genes and is mediated by a family of DNA methyltransferases (DNMTs). The loss of DNMTs results in genome-wide demethylation which causes alterations in gene expression. Recent studies showed that DNMT1, which maintains DNA methylation during DNA replication, localizes to UV laser-induced DNA damage areas. The aim of this study was to understand the mechanism of the recruitment of DNMT1 to areas of DNA damage and to investigate the function of DNMT1 in DNA damage repair pathways. Our preliminary results demonstrate that DNMT1 and PCNA localize to 800 nm laser-induced DNA damage, which induces DNA double strand breaks, by live cell imaging, but not other DNMTs like DNMT3A and DNMT3B. Catalytic subunit mutated (Cys1229Ala) DNMT1 also showed localization to DNA damage, but its recruitment was slow compared to wild type DNMT1. DNMT1 is targeted to sites of DNA replication via its PCNA binding domain (PBD) during S phase. To test whether PCNA is responsible for the recruitment of DNMT1 during DNA repair, we deleted the PBD (DNMT1 ΔPBD) and measured its localization. Unlike wild type DNMT1, DNMT1 ΔPBD remained diffuse and showed no accumulation at irradiated sites. 5-aza-2’-deoxycytidine (5-azadC) treatment, a drug which incorporates into DNA and covalently traps the DNMTs on DNA, results in the inhibition of DNMT1 recruitment to DNA damage areas, without affecting PCNA recruitment. To further investigate the mechanism of DNMT1 accumulation to DNA damage sites, we used drugs that inhibit known DNA damage repair proteins. After transfection of DNMT1 or PCNA, cells were treated with NU7026 (DNA-PKcs inhibitor), UCN-01 (CHK1 inhibitor) or caffeine (ATM and ATR inhibitor). DNMT1 and PCNA accumulation were not affected by NU7026 treatment, but UCN-01 and caffeine significantly inhibited both DNMT1 and PCNA accumulation to the irradiated region, indicating CHK1 activation is required for DNMT1/PCNA accumulation. Using immunofluorescence analyses after laser irradiation, we observed DNMT1 accumulation at DNA damage sites up to 2 hr after irradiation. Unlike DNMT1, PCNA accumulation was observed up to 6 hr after DNA damage in both S phase and non-S phase cells. To understand the role of DNMT1 in the DNA damage repair response, we used HCT116 cells deficient in DNMT1. Under normal conditions, γH2AX and phospho-ATM (pATM) levels, markers of DNA double strand breaks, were significantly increased in this mutant cell line. However, their recruitment to DNA damage sites was not changed compared to parental HCT116. Taken together our data suggest that CHK1 regulates the recruitment of DNMT1 and PCNA to DNA damage sites and DNMT1 is also involved in the regulation of DNA damage repair.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 485.