Functions of SRAP domain proteins in DNA metabolism

项目摘要 我们每个细胞中每天都有成千上万的无碱基位点形成。许多类型的环境毒素， DNA碱基的烷基化或氧化形成N7-鸟嘌呤加合物和8-氧代鸟嘌呤诱导脱碱基位点。 例如，存在于食品、洗涤剂、溶剂、塑料和农业中的N-亚硝胺 化学物质以及诸如四氯化碳、溴酸钾和氯仿等化学物质， 氧化应激都增加了DNA中脱碱基位点的频率。管理这种无处不在的 DNA损伤的形式可以导致包括癌症在内的多种疾病。已知的修复机制 需要一个完整的DNA双链体;然而，脱碱基位点在单链DNA中更容易形成， 是复制聚合酶的障碍。我们利用无偏见的蛋白质组学和遗传学方法， 了解复制叉如何应对基因毒性压力的挑战，前提是 在这两种方法中鉴定的未表征蛋白质将是新DNA损伤的强有力候选者 反应蛋白蛋白质组学和遗传学筛选均鉴定出HMCES（羟甲基胞嘧啶 胚胎细胞特异性）作为在复制叉处起作用的候选基因组维持蛋白。 据报道，HMCES可以结合并去除DNA中的5-羟甲基胞嘧啶，从而调节基因表达。 表情我们的初步数据表明，HMCES独立地发挥复制功能- 相关DNA修复蛋白。HMCES包含一个进化上古老的结构域（SRAP）。我们 假设SRAP蛋白提供了一种修复或耐受DNA损伤机制， 复制的这项提案将利用生物化学，遗传学和结构的方法在人类，酵母， 细菌系统，以确定SRAP蛋白如何维持基因组稳定性。完成这些研究 将在环境毒理学、DNA修复、DNA 复制、表观遗传控制和酶学。

PROJECT SUMMARY Thousands of abasic sites form daily in each of our cells. Many types of environmental toxins that cause alkylation or oxidation of DNA bases to form N7-guanine adducts and 8-oxoguanine induce abasic sites. For example, N-nitrosamines that are found in foods, detergents, solvents, plastics, and agricultural chemicals as well as chemicals like carbon tetracholoride, potassium bromate, and chloroform that induce oxidative stress all increase the frequency of abasic sites in DNA. Failures in managing this ubiquitous form of DNA damage can cause a variety of diseases including cancer. The known mechanisms of repair require an intact DNA duplex; however, abasic sites form more readily in single-stranded DNA where they are impediments to replicative polymerases. We utilized unbiased proteomic and genetic approaches to understand how replication forks deal with challenges to genotoxic stresses with the premise that uncharacterized proteins identified in both approaches would be strong candidates for new DNA damage response proteins. Both the proteomic and genetic screens identified HMCES (hydroxyl-methyl cytosine embryonic cell specific) as a candidate genome maintenance protein functioning at replication forks. HMCES was reported to bind and remove 5-hydroxymethyl cytosine from DNA and thereby regulate gene expression. Our preliminary data suggests that HMCES independently functions as a replication- associated DNA repair protein. HMCES contains an evolutionarily ancient domain (SRAP). We hypothesize that SRAP proteins provide a mechanism to repair or tolerate DNA damage during DNA replication. This proposal will utilize biochemical, genetic, and structural approaches in human, yeast, and bacterial systems to determine how SRAP proteins maintain genome stability. Completing these studies will generate paradigm setting discoveries within the fields of environmental toxicology, DNA repair, DNA replication, epigenetic control, and enzymology.