The MED1 Protein in DNA Damage Response and Repair

描述（由申请人提供）：遗传稳定性的维持不仅依赖于内源性和外源性DNA损伤的忠实修复，而且依赖于当损伤超过临界阈值时导致细胞周期停滞和细胞凋亡的检查点的迅速激活。 胞嘧啶和5-甲基胞嘧啶在CpG位点的自发脱氨为尿嘧啶和胸腺嘧啶是内源性突变的主要机制，并且在肿瘤发生中起重要作用。 几种尿嘧啶和胸腺嘧啶DNA N-糖基化酶对G：U和G：T错配有活性，存在于哺乳动物细胞中，并保护其免受CpG位点的脱氨基作用。 我们确定了这些酶之一，人MED 1（也称为MBD 4），作为错配修复（MMR）蛋白MLH 1的相互作用。灭活小鼠生殖系中的MED 1增加了CpG位点的致突变性，但仅增加了3倍，提高了通过备用修复活动进行补偿的可能性。 一种这样的候选物是具有与MED 1类似的体外生物化学活性的酶TDG。 此外，一般的MMR系统至少在原理上可以作用于G：T和G：U失配。 目前，MED 1、TDG和MMR在体内对CpG位点G：U和G：T错配修复的相对贡献尚不清楚。 最近，我们确定了一个新的作用，MED 1在DNA损伤反应的烷化剂和其他抗肿瘤药物。用增加剂量的烷化剂（如MNNG）和其他抗肿瘤药物处理的MED 1-/- MEFs未能经历细胞周期停滞和凋亡。 与MMR缺陷细胞非常相似，MED 1-/- MEFs对MNNG的抗性是由于耐受机制，因为DNA损伤累积但不引起G2-M检查点和p53激活。 在MED 1-/- MEFs中MMR蛋白水平显著降低，表明MED 1可能是通过维持MMR信号复合物的完整性来实现烷化剂细胞毒性所必需的。 我们假设MED 1在DNA修复和DNA损伤反应中具有双重作用。 本方案中的实验旨在：1）表征MED 1在DNA损伤诱导的G2-M期细胞周期阻滞和凋亡中的作用; 2）检查MED 1在维持MMR蛋白水平完整性中的作用; 3）检查MED 1、TDG和MMR在体内修复CpG位点G：T错配中的作用。 这些研究将为了解内源性突变、DNA损伤应答和肿瘤细胞抗化疗的机制提供新的见解。

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.