Abstract 1054: The role of DNMT1 in the DNA damage response

Abstract

Abstract DNA methyltransferases (DNMTs) are proteins reponsible for establishing and maintaining genome-wide patterns of DNA methylation, an epigenetic signature regulating gene expression and genome integrity. DNMT1 is regarded as the maintenance enzyme while DNMT3A/3B are involved in the de novo synthesis of DNA methylation pattern. Besides its known role in copying methylation during replication, DNMT1 has been reported to be involved in the DNA damage response by recruitment to damaged sites on DNA. However, the mechanism of how DNMT1 takes part in DNA damage repair (DDR) remains to be elucidated. Our previous study showed that DNMT1 is an early responder to DNA double-strand breaks and interacts with components of the DDR machinery (Ha, KS et al, 2011, Hum. Mol. Genet., 20 (1): 126-140). In order to further characterize the role of DNMT1 in DDR, we analyzed DNMT1 in cis and trans. In our previous study, we observed that DNMT1 is recruited to DNA damage sites rapidly and transiently. Since DNMT1 is present throughout the cell cycle, this rapid and transient localization suggests that, upon DNA damage, in order to localize on DNA damage sites, a fraction of the DNMT1 protein pool might be modified and/or its complement of binding partners transiently altered. To this end, we purified DNMT1 from mammalian cells before and after ionizing radiation and analyzed its post-translational modifications (PTMs) by mass spectrometry. DNMT1 shows multiple unique patterns of PTMs including acetylation and methylation before and after ionizing radiation. In order to characterize trans-acting partners of DNMT1 after DNA damage, we purified DNMT1 complexes after protein cross-linking and used mass spectrometry to determine the identity of interacting factors. Consistent with changes in the localization and PTM patterns on DNMT1 in the presence and absence of DNA damage, several known and novel DNMT1-protein interactions are also altered in cells with damaged DNA. Finally, in order to understand the role of DNMT1 in the DDR mechanism in a physiological context, we used siRNA to acutely reduce levels of DNMT1 in the presence and absence of DNA damage. Knock-down of DNMT1 reduced the activation of DDR signaling molecules such as ATM, CHK1, and γ-H2AX in a colon cancer cell line. Collectively, our results demonstrate that DNMT1 is a critical molecule involved in coordinating DNA damage signaling with DNA repair mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1054. doi:1538-7445.AM2012-1054