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。

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.