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

描述（由申请人提供）：Rad23和Rad4是DNA修复蛋白，在核苷酸切除修复（NER）中起作用。由Rad23和Rad4组成的复合物（称为NEF2）含有第三种称为Centrin的蛋白质（Cdc31），其在NER中的功能尚不清楚。Rad4和Centrin的表征是本研究的重点。Rad23通过阻止蛋白酶体降解Rad4来调节Rad4的稳定性。同样，Centrin增加了人类Xpc （Rad4同源物）的丰度。在酵母中，Centrin水平随着生长条件的变化而显著改变，Rad4丰度也随之变化。通过结合和调节Rad4的稳定性，Centrin可以影响NEF2复合物在DNA修复中的可用性。此外，Rad4本身可能会改变Centrin在细胞周期控制中的作用。中心蛋白通过促进纺锤杆体复制（SPB）来控制有丝分裂的生长，从而启动细胞周期的进入。我们的假设是，Centrin可以防止DNA损伤后的SPB复制，从而启动生长停滞并促进DNA修复。我们确定Centrin结合线粒体蛋白并影响ATP水平。与线粒体蛋白结合增加的Centrin突变体显示出更高的ATP水平。其中一个未能结合Rad4的突变体对紫外线敏感，揭示了线粒体功能与NER之间的潜在联系。建议进行以下遗传和生化研究。我们将确定i)酵母Centrin是否稳定Rad4 ii)，以及这种相互作用是否受到DNA损伤的影响。iii) Rad4的稳定性也受到Rad23的调控，并提出了Rad23/Rad4相互作用的研究。iv)将检测Centrin突变蛋白与Rad4的相互作用、NEF2的组装以及与线粒体蛋白的结合。v) DNA损伤后，在突变体Centrin和Rad4蛋白存在的情况下，梭形极体的亚细胞分布和复制将被表征。核苷酸切除修复通路的缺陷可引起色素性干皮病，这是一种易患癌症的疾病，可导致早期死亡。确定Centrin的作用将使我们能够确定生长控制机制如何促进有效的DNA修复。有缺陷的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，是美国人口中发病率增长最快的疾病之一。核苷酸切除修复在清除日光-光诱导的DNA损伤中起着核心作用，Rad4是这种细胞防御机制中的一个重要蛋白质。然而，Rad4的功能还没有被很好地理解，它的特性是本应用程序的重点。

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.