REPAIR OF DNA DAMAGED BY UV IRRADIATION IN YEAST

该项目的长期目标是确定真核细胞复制受损DNA的机制。特别是，将研究rad6 - rad18依赖于紫外损伤DNA的无错误和易错误旁路复制的遗传控制和分子机制。色素性干皮病（XP）患者对阳光敏感，他们患皮肤癌的几率很高。XP可以由核苷酸切除修复缺陷引起，或者，在XP的变体形式中，由紫外线损伤DNA的无错误复制缺陷引起。易出错的紫外线病变复制旁路导致XP细胞突变性增加，这是XP患者癌症发病率升高的根本原因。拟议的研究将通过遗传学手段鉴定MMS2-UBC13依赖性无错误复制后修复的其他成分。Rad5与PCNA相互作用的生物学和功能意义将通过遗传和生化手段进行研究。Rad5在DNA解绕和D环形成中的作用将被研究，Rad5依赖的DNA合成“拷贝选择”模式将用纯化的蛋白质重建。rad30编码的DNA聚合酶eta对未受损DNA的处理能力和保真度将被确定，遗传和生化研究将检查Pol-eta绕过顺式同步T-T二聚体和（6-4）光产物的能力。rad30的3‘- bbb50 ’外切酶活性将被表征，并确定其在校对中的作用。在rad30中保守的酸性残基在其DNA聚合酶和3‘- bbb50 ’外切酶活性中的作用将被研究。用高纯度的组分重组Rev3-Rev7依赖性T-T二聚体和（6-4）光产物的诱变转译合成，并分析Rev1和其他蛋白如PCNA和RFC在这一过程中的作用。鉴定DNA复制/损伤旁路蛋白在DNA损伤反应中泛素化，并确定RAD6和RAD18在这一过程中的作用。

The long term objectives of this project are to define the mechanisms that eukaryotic cells employ to replicate damaged DNA. In particular, the genetic control and molecular mechanisms of Rad6-Rad 18 dependent error- free and error-prone bypass replication of UV damaged DNA will be studied in the yeast Saccharomyces cerevisiae. Xeroderma pigmentosum (XP) patients are sensitive to sunlight and they suffer from a high incidence of skin cancers. XP can arise from a defect in nucleotide excision repair, or, in the variant form of XP, from a defect in error-free replication of UV damaged DNA. Error-prone replicative bypass of UV lesions leads to increased mutagenicity in XP cells, the underlying cause of elevated cancer frequency in XP patients. The proposed studies will identify by genetic means the other components of the MMS2-UBC13 dependent error-free postreplicational repair. The biological and functional significance of Rad5 interaction with PCNA will be examined by genetic and biochemical means. The role of Rad5 in DNA unwinding and in D loop formation will be examined, and Rad5-dependent "copy choice" mode of DNA synthesis will be reconstituted with purified proteins. The processivity and fidelity of RAD3O encoded DNA polymerase eta on undamaged DNA will be determined, and genetic and biochemical studies will examine the ability of Pol-eta to bypass a cis-syn T-T dimer and a (6-4) photoproduct. The 3'-> 5' exonuclease activity of Rad 30 will be characterized, and its role in proofreading determined. The role of conserved acidic residues in Rad3O in its DNA polymerase and 3'-> 5' exonuclease activities will be investigated. The Rev3-Rev7 dependent mutagenic translesion synthesis of T-T dimer and of (6-4) photoproduct will be reconstituted with highly purified components, and the role of Rev1 and other proteins such as PCNA and RFC in this process will be analyzed. The DNA replication/damage bypass proteins which become ubiquitinated in response to DNA damage will be identified, and the role of RAD6 and RAD18 in this process will be determined.