芽殖酵母RAD5及相关基因在DNA损伤耐受中的作⽤机制

DNA damage tolerance (DDT) functions to bypass replication-blocking lesions and is subdivided into two parallel pathways: error-prone translesion DNA synthesis (TLS) and error-free DDT. In budding yeast, the Rad6-Rad18 complex monoubiquitinates PCNA(Pol30) at the K164 residue to promote TLS, while the Mms2-Ubc13-Rad5 complex is required for the K63-linked PCNA polyubiquitination at the same K164 residue. In addition, the same K164 residue can be sumoylated by Siz1-Ubc9, which leads to recruitment of an Srs2 helicase to prevent homologous recombination. Based on our observations and literature, we hypothesize that the RAD5 function is not limited to error-free DDT ad may play a regulatory role in all three DDT branches. As the first step to test this hypothesis, we demonstrated that Rad5 is involved in TLS through physical interaction with a TLS polymerase Rev1. In this proposal, we plan to systematically evaluate additional functions of RAD5 and related genes to further understand the DDT mechanism in budding yeast. Firstly, we will examine the physical interaction between Rad5 and PCNA and determine the biological significance of this interaction. Secondly, we have discovered a PCNA ubiquitination-independent but Pol30-K164 dependent function of Rad5, pointing to its involvement in the pathway mediated by Pol30-K164 sumoylation. We will reveal the underlying molecular mechanism of this involvement through its possible interactions with Ubc9, SUMO and Srs2. Thirdly, we will address whether and how the Shu and Rad55-Rad57 complexes are involved in coordinating PCNA promoted, Srs2-mediated inhibition of HR. The above studies will significantly advance our current knowledge of eukaryotic DDT. Finally, as a long-term objective, we plan to perform a synthetic genetic array with DDT genes as baits to search for novel genes involved in yeast DDT. Since all genes involved in DDT known to date are highly conserved throughout eukaryotes, from yeast to plants and human, this study is expected to impact on agriculture and public health.

DNA损伤耐受能够通过两条平行⽀路跨过阻⽌DNA 复制的损伤，即易错的跨损伤合成和无错的耐受途径。在酵母中，Rad6/18 复合体将 PCNA-K164位点单泛素化，从而促进跨损伤合成，⽽含 Rad5 的复合体以K63链的方式将PCNA 多泛素化从而促进无错耐受途径。此外，K164位点还可以通过被SUMO化来招募Srs2解旋酶从而抑制不必要的同源重组。基于前期研究，我们认为 RAD5的功能不仅局限于无错耐受通路，因此计划系统地研究 RAD5 额外功能及其相关基因，进而加深对耐受机制的了解以期对 Rad5 功能进⾏重新评估。这些潜在的功能包括参与跨损伤合成、影响 PCNA调控及其 SUMO 化等。 上述研究将拓展对真核生物耐受途径的认知。长远来看，我们还将通过合成遗传阵列分析进一步寻找参与酵母耐受途径的新基因。鉴于前述相关基因在真核生物中都高度保守，本研究将对农业和公共卫生方面产生积极影响。

本课题重点关注芽殖酵母DNA损伤耐受的机理和途径选择。芽殖酵母的DNA损伤耐受途径主要分为跨损伤合成和无错复制，其中PCNA的翻译后修饰发挥重要作用。PCNA的单泛素化修饰介导跨损伤合成，在此基础上的多聚泛素化修饰则介导无错复制。通过对PCNA翻译后修饰的一系列参与因子，例如Rad5和Rad18的研究，以及对参与两条途径的因子，例如Rev1、Polη和Sgs1的研究能够帮助我们更加深入地了解芽殖酵母的DNA损伤耐受途径。我们在此资助周期的主要研究成果有：第一，我们发现Rad5能够识别特定的DNA结构从而介导芽殖酵母进入跨损伤合成途径。第二，我们发现Sgs1参与到了芽殖酵母的两条DNA损伤耐受途径中，并有可能在修复途径的选择上发挥重要作用。第三，我们发现Rev1和Polη在应对不同类型的DNA损伤的过程中发挥不同的功能。我们的研究更加深入地揭示了芽殖酵母DNA损伤耐受途径的机理，为研究芽殖酵母DNA损伤耐受途径的选择机制提供了依据。在研究的过程中，我们构建了一些高效、简便、实用的基因改造技术，这些技术有的是关于在染色体中为一个目标位点插入标签，有的是关于在染色体上置换内源的启动子，有的是关于如何研究一个必须基因。我们所研究的技术有的解决了以往类似的基因改造技术中的不足，让这类技术变得更加可靠；有的解决了这个领域研究的难题，让本来很难完成的研究变得可能。我们提出的这些技术为芽殖酵母中的研究带来了巨大便利，相信未来会被相关领域的研究人员广泛采用。