【作者】 毕晓辉；

【导师】 郝水； 黄百渠；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2002， 博士

【摘要】 泛素系统通过对细胞内短期存在蛋白的及时降解而在各项生命活动中起着重要作用。在泛素系统中起主要作用的是三种酶：泛素激活酶E1；泛素缀合酶E2/Ubc和泛素连接酶E3。这三种酶协同作用把泛素分子链作为标签连接在目的蛋白上使之被26S蛋白酶体识别而降解。在DNA损伤修复中，几种泛素系统中的酶起着至关重要的作用。其中复制后修复(PRR)主要是由RAD6上位体系负责的。这一体系的主要成员为Rad6，Rad18，Rad5和Mms2-Ubc13异二聚体。Rad6Ubc2和Ubc13是两种Ubc，Mms2是一种Ubc类似蛋白。Rad18是一种单链DNA结合蛋白，具有许多泛素系统E3所有的典型RING结构，与Rad6(Ubc2)形成结合紧密的异二聚体。Rad5同Rad18一样是具有RING结构的单链结合蛋白，且通过其RING结构与Mms2-Ubc13异二聚体结合。在DNA修复过程中，Rad18和Rad5互相结合并结合于DNA上，这样就把Rad6和Mms2-Ubc13同时招募于DNA损伤部分。但是具体这一修复复合物的泛素化底物蛋白是什么，它们又具体结合于什么样性质的DNA损伤部分，目前并不清楚，而这两点是研究泛素系统在DNA修复中的作用的关键。 鉴于Rad18在此复合物中起媒介Ubc和DNA作用，同时它本身又可能是一种E3，我们便从Rad18与其它非PRR路径修复的DNA损伤结构的相互关系入手，来探讨泛素系统在其它DNA修复路径中是否也会起作用。利用染色质免疫沉淀技术，我们首次发现了Rad18结合于DNA双链损伤(DSB)的两端。这无疑表明Rad18在DNA各种损伤修复中作用的广泛性。虽然这一发现只是我们工作的一个开端，但是基于这一开端，两个层面的研究工作将被很大程度地拓宽，一是泛素系统不仅只是在DNA修复的PRR路径起作用，而是可能涉及DNA修复的各种不同路径。二是DSB修复的路径中可能又多了一种修复因子Rad18，它与已知的Rad52上位修复体系的关系，以及Rad6等PRR路径中的其它蛋白与已知DSB修复体系的关系将被人们所关注。更多还原

【Abstract】 In the DNA postreplication repair (PRR) RAD6 epitasis pathway, the error-free group comprises RAD 18, RAD5 and the MMS2-UBC13 heterodimer factors. Both Rad6 and UbclS are ubiquitin-conjugating enzymes, and Mms2 is a UBC-like protein. RadlS and Rad5 are both single strand binding RING linger proteins. RAD6 binds to RadlS and Mms2-UbclS binds to Rad5. The interaction between RadlS and Rad5 mediates the cooperation between the Ubcs, and also localizes the Ubcs on DNA. However, the ubiquitylated substrate protein of these Ubcs remains unknown, and the nature of the DNA to which the repair factors bind is still unclear. So the main objective of my PhD work is to investigate the role played by the ubiquitin system during DNA repair. We intended to use RadlS as a key element to start the work, as RadlS interacts with both Ubc and DNA. We intended to investigate what types of DNA structures RadlS is associated with in vivo.The DNA double strand break (DSB) can be rejoined by at least two discrete pathways, i.e., the homologous recombination and the nonhomologous end-joining. At least 10 genes were found involved in NHEJ. Among them, Yku70 (Hdfl) and YkuSO (Hdf2) are break ends binding proteins, and RAD52, MRE11, XRS2 are found both in HR and NHEJ. In Saccharomyces cerevisiae, switching of mating-type is associated with a DSB, created by the site-specific endonuclease HO. It is reported that RAD 18 is found to be involved in Ho degradation via the ubiquitin 26S proteasome system. RAD5 is found involved in the avoidance of non-homologous end-joining of DSB in S. cerevisiae. Therefore, our hypothesis is that RadlS may directly associate with DSB. And also for the establishment of the technique, we were interested in examining whether RAD18 plays a role in this HO-induced DSB repair in S. cerevisiae.To measure the association of RadlS with DSB, we used the chromatin immunoprecipitation (CHIP). In this technique, first the specific protein-DNA interaction is crosslinked by formaldehyde, and the proteins bound with the specific DNA are immunoprecipitated by antibodies. Then the precipitated DNA is purified by reversing theprotein-DNA with 1% SDS and heat In our experiment, RAD 18 was tagged with 9myc in vivo. We introduced the DSB by the induction of HO expression, which is under the control of the Gal 4 Promoter. Shortly after DSB induction, CHIP was performed with anti-myc antibodies. For multiplex PCR, primers specific for Z and Y a, which are involved in the DSB, were employed. And also, a pair of primers of ACT1 was used as a negative control.In this experiment, we also used Yku70 as a positive control, since it has been well defined as a DSB end binding protein. We found RadlS directly binds to the two ends of DSB. The results suggested that RadlS plays a role in DSB repair, as well as in PRR. This work is also suggestive of the discovery of a novel DSB repair factor.更多还原