Functions of SRAP domain proteins in DNA metabolism

项目总结 在我们每个细胞中，每天都有成千上万个基本部位形成。许多类型的环境毒素会导致 DNA碱基的烷基化或氧化形成N7-鸟嘌呤加合物和8-氧鸟嘌呤可产生碱性位点。 例如，在食品、洗涤剂、溶剂、塑料和农产品中发现的N-亚硝胺 化学品以及四氯化碳、溴酸钾和氯仿等化学品会导致 氧化应激都会增加DNA中基本位点的频率。在管理这种无处不在的问题上失败 DNA损伤的形式会导致包括癌症在内的多种疾病。已知的修复机制 需要完整的DNA双链；然而，在单链DNA中更容易形成基本位点，在那里它们 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 了解复制分叉如何应对基因毒性压力的挑战，前提是 在这两种方法中发现的未鉴定蛋白质将是新的dna损伤的有力候选者。 反应蛋白。蛋白质组和遗传筛选都鉴定了HMCES(羟甲基胞嘧啶 胚胎细胞特异性)作为在复制分叉起作用的候选基因组维持蛋白。 据报道，HMCES可以结合和去除DNA中的5-羟甲基胞嘧啶，从而调节基因 表情。我们的初步数据表明，HMCEs独立地发挥着复制功能- 相关的DNA修复蛋白。HMCES包含一个进化古域(SRAP)。我们 假设SRAP蛋白提供了一种修复或耐受DNA损伤的机制 复制。这项建议将利用生化、遗传和结构方法在人类、酵母和 确定SRAP蛋白如何维持基因组稳定性的细菌系统。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.