Function of NEF4 in Nucleotide Excision Repair

描述(申请人提供)：修复受损DNA的主要途径之一是通过核苷酸切除修复(NER)。人类NER的缺陷将NER修复受损的DNA与癌症的易感性联系在一起。NER反应在物种之间是保守的，我们利用酿酒酵母的优势来定义NER复合体NEF4的作用。NEF4是酵母中有效修复非转录DNA所必需的。NEF4复合体由三种蛋白质组成：Rad16、Rad7和Elc1。Rad16含有一个Snf2/Swi2ATPase结构域，可能被认为与扫描DNA损伤有关。最近，我们发现NEF4含有类似于其他E3泛素连接酶复合体的保守基序。这些结果将泛素介导的一条新的NER通路与参与DNA损伤识别的蛋白质Rad4的调节联系在一起。通过体外和体内NER实验，我们将确定NEF4 ATPase和泛素连接酶活性的功能。此外，染色质免疫沉淀(ChIP)分析将使我们能够确定NEF4在调节和招募其他NER组分到DNA受损部位的作用。

DESCRIPTION (provided by applicant): One of the main pathways for repair of damaged DNA is through Nucleotide Excision Repair (NER). Defects in NER in humans have linked the repair of damaged DNA by NER to the predisposition to cancer. The NER reaction is conserved across species and we have exploited the advantages of S. cerevisiae to define the role of the NER complex NEF4. NEF4 is required for the efficient repair of nontranscribed DNA in yeast. The NEF4 complex consists of three proteins: Rad16, Rad7, and Elc1. Rad16 contains an Snf2/Swi2 ATPase domain perhaps thought to be involved in scanning the DNA for damage. More recently, we have identified NEF4 as containing conserved motifs similar to other E3 ubiquitin ligase complexes. These results link a novel ubiquitin-mediated pathway to NER and the regulation of Rad4, a protein involved in DNA damage recognition. Through the use of in vitro and in vivo NER assays, we will define the function of the NEF4 ATPase and ubiquitin ligase activities. Additionally, Chromatin immunoprecipitation (ChIP) assays will allow us to define the role of NEF4 in regulation and recruitment of other NER components to the sites of damaged DNA.