"Studies to examine Centrin's role in DNA repair"

描述（由申请人提供）：Rad 23和Rad 4是在核苷酸切除修复（NER）中起作用的DNA修复蛋白。由Rad 23和Rad 4组成的复合物（称为NEF 2）含有第三种蛋白质，称为中心蛋白（Cdc 31），其在NER中的功能尚不清楚。Rad 4和Centrin的特性是本研究的重点。Rad 23通过防止蛋白酶体降解Rad 4来调节Rad 4的稳定性。类似地，中心蛋白增加人Xpc（Rad 4直系同源物）的丰度。在酵母中心蛋白水平显着改变的生长条件，并在Rad 4丰度的一致变化进行了观察。通过结合和调节Rad 4的稳定性，中心蛋白可以影响NEF 2复合物用于DNA修复的可用性。此外，Rad 4本身可能改变Centrin在细胞周期控制中的作用。中心蛋白通过促进纺锤体-极体复制（SPB）来控制有丝分裂生长，SPB启动细胞周期进入。我们的假设是，中心蛋白阻止SPB复制后，DNA损伤启动生长停滞和促进DNA修复。我们确定中心蛋白结合线粒体蛋白并影响ATP水平。与线粒体蛋白结合增加的中心蛋白突变体显示出更高的ATP水平。一个这样的突变体，未能结合Rad 4是敏感的紫外线，揭示了线粒体功能和NER之间的潜在联系。建议进行以下遗传和生物化学研究。我们将确定i）酵母中心蛋白是否稳定Rad 4 ii），以及这种相互作用是否受到DNA损伤的影响。iii）Rad 4稳定性也受Rad 23调节，并且提出研究以检查Rad 23/Rad 4相互作用。iv）将检查中心蛋白突变体蛋白与Rad 4的相互作用、NEF 2的组装以及与线粒体蛋白的结合。v）在DNA损伤后，在突变的中心蛋白和Rad 4蛋白存在下，将表征中心蛋白的亚细胞分布和纺锤体极体的复制。核苷酸切除修复途径的缺陷可导致着色性干皮病，这是一种易患癌症的疾病，可导致早死。确定中心蛋白的作用将使我们能够确定生长控制机制如何有助于有效的DNA修复。缺陷DNA修复和未能阻止细胞生长是基因组不稳定的主要原因，并且在许多形式的恶性生长中观察到。皮肤癌是一个重要的健康问题，并且是美国人口中发病率增长最快的疾病之一。核苷酸切除修复在去除日光诱导的DNA损伤中起着核心作用，并且在这种细胞防御机制中的重要蛋白是Rad 4。然而，Rad 4的功能还没有得到很好的理解，其表征是本申请的重点。

DESCRIPTION (provided by applicant): Rad23 and Rad4 are DNA repair proteins that function in nucleotide excision-repair (NER). A complex consisting of Rad23 and Rad4 (termed NEF2), contains a third protein called Centrin (Cdc31), whose function in NER is unknown. The characterization of Rad4 and Centrin is the focus of this investigation. Rad23 regulates Rad4 stability by preventing its degradation by the proteasome. Similarly, Centrin increases the abundance of human Xpc (Rad4 ortholog). In yeast Centrin levels were dramatically altered by growth conditions, and a coincident change in Rad4 abundance was observed. By binding and regulating the stability of Rad4, Centrin could influence the availability of the NEF2 complex for DNA repair. Furthermore, Rad4 might itself alter Centrin's role in cell-cycle control. Centrin controls mitotic growth by promoting spindle-pole body duplication (SPB), which initiates cell cycle entry. Our hypothesis is that Centrin prevents SPB duplication following DNA damage to initiate growth arrest and promote DNA repair. We determined that Centrin binds mitochondrial proteins and influences ATP levels. A Centrin mutant with increased binding to mitochondrial proteins showed higher ATP levels. One such mutant that failed to bind Rad4 was sensitive to UV light, revealing a potential link between mitochondrial function and NER. The following genetic and biochemical studies are proposed. We will determine if i) yeast Centrin stabilizes Rad4 ii), and if this interaction is affected by DNA damage. iii) Rad4 stability is also regulated by Rad23, and studies are proposed to examine Rad23/Rad4 interaction. iv) The interaction of Centrin mutant proteins with Rad4, assembly of NEF2, and binding to mitochondrial proteins will be examined. v) The subcellular distribution of Centrin and duplication of the spindle pole-body will be characterized after DNA damage, and in the presence of mutant Centrin and Rad4 proteins. Defects in the nucleotide excision repair pathway can cause xeroderma pigmentosum, a cancer- prone condition that can lead to early death. Defining the role of Centrin will allow us to determine how growth control mechanisms contribute to efficient DNA repair. Defective DNA repair and failure to arrest cell growth is a major cause of genome instability, and is observed in many forms of malignant growth. Skin cancer is an important health concern, and shows one of the fastest incidences of increase in the US population. Nucleotide excision repair plays a central role in the removal of sunlight-light induced DNA damage, and an important protein in this cellular defense mechanism is Rad4. However, the function of Rad4 is not well understood, and its characterization is the focus of this application.