DNA REPAIR SIGNALING BY NOVEL POLYUBIQUITIN CHAINS

通过Ub的赖氨酸-48（K48）连接的多聚泛素（polyUb）链代表Ub-蛋白酶体蛋白水解途径中的主要靶向信号，而通过K63连接的polyUb链通过似乎不涉及蛋白水解的机制在复制后DNA修复中起作用。这些观察结果表明，Ub组装成不同类型的聚合物可能是一种机制，以多样化的信号功能的Ub。为了验证这一假设，我们将应用生物化学和酵母分子遗传学的方法来表征K63连接的polyUb链的组装和识别，并阐明这些聚合物在DNA修复中的信号功能。我们发现酵母酶在体外组装均聚K63连接的polyUb链，并在体内DNA修复中发挥作用，为这项工作提供了起点。这些蛋白是Ubcl 3 p（Ub缀合酶）和Mms 2 p（Ub E2变体或UEV蛋白）。通过描述Mms 2 p/Ubc 13 p复合物的机制和功能，我们将阐明信号产生的过程;通过识别与K63连接链相连的细胞蛋白质，我们将深入了解信号识别的功能后果;最后，通过使用体外组装的链作为工具，我们将直接解决K63连接的polyUb链的蛋白水解信号传导能力，并分离可能代表所提出的DNA修复信号通路中下游元件的链识别因子。这些研究的结果将为Ub在DNA修复中的机制作用提供新的见解，并可能最终导致改善癌症和其他疾病的治疗策略。此外，如果我们的研究结果支持polyUb链用于多样化Ub信号功能的假设，则将有助于解释这种小分子如何能够在多个细胞过程中充当不同的信号。最后，我们的研究结果将提供第一个生化洞察UEV蛋白家族的功能，在哺乳动物中包括肿瘤抑制因子Tsgl 01，以及其他与分化和细胞转化有关的蛋白。

Polyubiquitin (polyUb) chains linked through lysine-48 (K48) of Ub represent the predominant targeting signal in the Ub-proteasome proteolytic pathway, whereas polyUb chains linked through K63 function in postreplicative DNA repair by a mechanism which does not appear to involve proteolysis. These observations suggest that the assembly of Ub into distinct types of polymers may be a mechanism to diversify the signaling functions of Ub. To test this hypothesis we will apply methods of biochemistry and yeast molecular genetics to characterize the assembly and recognition of K63-linked polyUb chains, and elucidate the signaling function of these polymers in DNA repair. Our discovery of yeast enzymes which assemble homopolymeric K63-linked polyUb chains in vitro, and function in DNA repair in vivo, provides the starting point for this work. These proteins are Ubcl3p (a Ub conjugating enzyme) and Mms2p (a Ub E2 variant or UEV protein). By characterizing the mechanism and function of the Mms2p/Ubc13p complex, we will explicate the process of signal generation; by identifying cellular proteins that are linked to K63-linked chains, we will gain insight into the functional consequences of signal recognition; finally, by using an in vitro-assembled chain as a tool, we will directly address the proteolytic signaling competence of K63-linked polyUb chains, and isolate chain-recognizing factors that may represent downstream elements in the proposed DNA repair signaling pathway. The results of these studies will provide new insights into the mechanistic role of Ub in DNA repair, and may ultimately lead to improved strategies for the treatment of cancer and other diseases. Moreover, if our results support the hypothesis that polyUb chains serve to diversify the signaling functions of Ub, it will help to explain how this small molecule is able to act as a distinct signal in multiple cellular processes. Finally, our results will provide the first biochemical insight into the functioning of UEV protein family, which in mammals includes the tumor suppressor Tsgl0l, and other proteins implicated in differention and cell transformation.