DNA REPAIR SIGNALING BY NOVEL POLYUBIQUITIN CHAINS

通过Ub的赖氨酸-48(K48)连接的多聚泛素(PolyUb)链是Ub-蛋白酶体蛋白分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分解的机制在复制后DNA修复中发挥作用。这些观察结果表明，将Ub组装成不同类型的聚合物可能是使Ub信号功能多样化的一种机制。为了验证这一假说，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。我们发现的酵母酶可以在体外组装K63连接的多聚Ub链，并在体内发挥DNA修复功能，为这项工作提供了起点。这些蛋白是Ubcl3p(一种Ub结合酶)和Mms2p(一种Ub E2变异体或UEV蛋白)。通过对Mms2p/Ubc13p复合体的机制和功能的研究，我们将阐明信号产生的过程；通过鉴定与K63连锁的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，我们将直接研究K63连锁的PolyUb链的蛋白水解性信号功能，并分离可能代表所提出的DNA修复信号通路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，并最终可能导致改善癌症和其他疾病的治疗策略。此外，如果我们的结果支持PolyUb链用于使Ub的信号功能多样化的假设，这将有助于解释这种小分子如何能够在多种细胞过程中扮演不同的信号角色。最后，我们的结果将首次提供对UEV蛋白家族功能的生化洞察，其中包括哺乳动物中的肿瘤抑制基因Tsgl01，以及与分化和细胞转化有关的其他蛋白质。

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.