The MED1 Protein in DNA Damage Response and Repair

描述(申请人提供)：保持遗传稳定性不仅依赖于对内源性和外源性DNA损伤的忠实修复，还依赖于当损伤超过临界阈值时，迅速激活检查点，导致细胞周期停滞和细胞凋亡。胞嘧啶和5-甲基胞嘧啶在CpG位点自发脱氨为尿嘧啶和胸腺嘧啶是内源性诱变的主要机制，在肿瘤发生中起重要作用。几种尿嘧啶和胸腺嘧啶DNA N-糖基酶在哺乳动物细胞中存在G：U和G：T错配，并保护其免受CpG位点脱氨事件的影响。我们发现其中一种酶，人MED1(也称为MBD4)，是错配修复(MMR)蛋白MLH1的相互作用或相互作用。小鼠生殖系中灭活的MED1增加了CpG位点的突变性，但仅增加了3倍，增加了通过备份修复活动进行补偿的可能性。其中一个候选是TDG酶，它在体外具有类似于MED1的生化活性。此外，至少在原则上，一般的MMR系统可以作用于G：T和G：U失配。目前，MED1、TdG和MMR在体内修复CpG位点G：U和G：T错配中的相对作用尚不清楚。最近，我们发现了MED1在烷化剂和其他抗肿瘤药物对DNA损伤反应中的一个新的作用。用增加剂量的烷化剂，如MNNG和其他抗肿瘤药物治疗的MED1-/-MEF，未能经历细胞周期停滞和细胞凋亡。就像MMR缺陷细胞一样，MED1-/-MEF对MNNG的耐受是由于耐受机制，因为DNA损伤累积，但不会引发G2-M检查点和P53激活。在MED1-/-MEF中，MMR蛋白水平显著降低，这表明MED1可能是通过维持MMR信号复合体的完整性来实现烷化剂细胞毒性所必需的。我们假设MED1在DNA修复和DNA损伤反应中具有双重作用。该方案中的实验旨在：1)研究MED1在DNA损伤诱导的G2-M细胞周期停滞和凋亡中的作用；2)研究MED1在维持MMR蛋白水平完整性中的作用；3)研究MED1、TDG和MMR在体内修复CpG位点G：T错配中的作用。这些研究将为内源性诱变、对DNA损伤的反应以及癌细胞对抗肿瘤化疗的耐药性提供新的见解。

DESCRIPTION (provided by applicant): Maintenance of genetic stability relies not only on faithful repair of endogenous and exogenous DNA damage, but also on the prompt activation of checkpoints leading to cell cycle arrest and apoptosis, when damage exceeds a critical threshold. Spontaneous deamination of cytosine and 5-methylcytosine to uracil and thymine at CpG sites is a major mechanism of endogenous mutagenesis and plays an important role in tumorigenesis. Several uracil and thymine DNA N-glycosylases active on G: U and G: T mismatches are present in mammalian cells and protect from deamination events at CpG sites. We identified one of these enzymes, human MED1 (also known as MBD4), as an interact or of the mismatch repair (MMR) protein MLH1. Inactivation MED1 in the mouse germ line increases mutagenicity at CpG sites, but only 3-fold, raising the possibility of compensation by back-up repair activities. One such candidate is the enzyme TDG that has in vitro biochemical activities similar to MED1. Furthermore, the general MMR system may, at least in principle, act on G: T and G: U mismatches. At the moment, the relative contributions of MED1, TDG and MMR to the repair of G: U and G: T mismatches at CpG sites in vivo are unclear. Recently, we identified a novel role of MED1 in the DNA damage response to alkylating agents and other anti-tumor drugs. MED1-/- MEFs treated with increasing doses of alkylating agents, like MNNG, and other anti-tumor drugs, failed to undergo cell cycle arrest and apoptosis. Much like MMR-defective cells, resistance of MED1-/- MEFs to MNNG was due to a tolerance mechanism, as DNA damage accumulated but did not elicit a G2-M checkpoint and p53 activation. MMR proteins levels are markedly reduced in MED1-/- MEFs, suggesting that MED1 may be required for alkylating agent cytotoxicity by maintaining integrity of the MMR signaling complex. We hypothesize that MED1 has a dual role in DNA repair and DNA damage response. Experiments in this proposal are designed to: 1) characterize the role of MED1 in G2-M cell cycle arrest and apoptosis induced by DNA damage; 2) examine the role of MED1 in maintaining integrity of MMR protein levels; 3) examine the roles of MED1, TDG, and MMR in the repair of G: T mismatches at CpG sites in vivo. These studies will provide new insights into mechanisms of endogenous mutagenesis, response to DNA damage and resistance of cancer cells to anti-tumor chemotherapy.