RAD5 INTERACTING PROTEIN SEARCH BY YEAST TWO HYBRID SCREENING

通过酵母二杂交筛选 RAD5 相互作用蛋白

• 批准号: 7420761
• 负责人: Kyungjae Myung
• 金额: $ 0.5万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-20 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7420761
• 关键词: DNA damage; Saccharomyces cerevisiae; cell cycle; proteins; suppression; ubiquitin; yeasts

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Persistent stalled replication forks collapse and cause genomic instability (Kolodner et al., 2002). In yeast, stalled replication forks are resolved either by bypassing DNA damage with translesion synthesis polymerases or by template-switching to the nascent strand of sister chromatid (Smirnova and Klein, 2003; Ulrich, 2005). The decision of these two pathways is regulated through the modification of Proliferating Cell Nuclear Antigen (PCNA). PCNA is monoubiquitinated by Rad18 and further poly-ubiquitinated by Rad5 on the monoubiquitinated PCNA (Ulrich, 2005). We found that Rad18 and Rad5 suppress gross chromosomal rearrangement (GCR) through the poly-ubiquitination of PCNA (Motegi et al., 2006). This suppression mechanism is linked with the cell cycle checkpoint pathway. In this study, we also demonstrated that another Small Ubiquitin-like Modifier (SUMO) ligase Siz1 functions antagonistically by modifying PCNA to promote GCR formation through its interaction with a helicase Srs2 and the Rad51-dependent recombination machinery. Our data place Rad5 and Rad18 in the cell cycle checkpoint dependent GCR suppression pathway and added the Siz1-Srs2 directed Rad51 dependent recombination pathway as a third GCR enhancing pathway. To study mechanisms how the Rad5 pathway directs DNA damage avoidance pathway by template-switching, we set up a collaboration with Marrissa Vignali (U. Washington) to find yeast Rad5 interacting factors by yeast two-hybrid screening. We will screen entire yeast open reading frames in normal and in DNA damage conditions. Genes encoding proteins identified from these screenings will be tested whether they function to suppress GCR formation. References) Kolodner, R.D., C.D. Putnam, and K. Myung. 2002. Maintenance of genome stability in Saccharomyces cerevisiae. Science. 297:552-7. Motegi, A., K. Kuntz, A. Majeed, S. Smith, and K. Myung. 2006. Regulation of gross chromosomal rearrangements by ubiquitin and SUMO ligases in Saccharomyces cerevisiae. Mol Cell Biol. 26:1424-33. Smirnova, M., and H.L. Klein. 2003. Role of the error-free damage bypass postreplication repair pathway in the maintenance of genomic stability. Mutat Res. 532:117-35. Ulrich, H.D. 2005. The RAD6 pathway: control of DNA damage bypass and mutagenesis by ubiquitin and SUMO. Chembiochem. 6:1735-43.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。持续停滞的复制叉崩溃并导致基因组不稳定性（Kolodner等人，2002年）。在酵母中，停滞的复制叉通过用translesion合成聚合酶绕过DNA损伤或通过模板转换到姐妹染色单体的新生链来解决（Smirnova和Klein，2003; Ulrich，2005）。这两条通路的决定是通过修饰增殖细胞核抗原（PCNA）来调节的。PCNA被Rad 18单泛素化，并在单泛素化的PCNA上进一步被Rad 5多泛素化（Ulrich，2005）。我们发现Rad 18和Rad 5通过PCNA的多泛素化抑制总染色体重排（GCR）（莫泰吉et al.，2006年）。这种抑制机制与细胞周期检查点途径有关。在这项研究中，我们还表明，另一个小泛素样修饰剂（SUMO）连接酶Siz 1的功能拮抗性修改PCNA，促进GCR的形成，通过其与解旋酶Srs 2和Rad 51依赖性重组机制的相互作用。我们的数据将Rad 5和Rad 18置于细胞周期检查点依赖性GCR抑制途径中，并添加了Siz 1-Srs 2指导的Rad 51依赖性重组途径作为第三种GCR增强途径。为了研究Rad 5通路如何通过模板转换指导DNA损伤避免通路的机制，我们与Marrissa Vignali（U。华盛顿）通过酵母双杂交筛选寻找酵母Rad 5相互作用因子。我们将在正常和DNA损伤条件下筛选整个酵母开放阅读框。将测试从这些筛选中鉴定的编码蛋白质的基因是否具有抑制GCR形成的功能。 参考文献）Kolodner，R.D.，C.D. Putnam和K.明哥2002.酿酒酵母基因组稳定性的维持。科学297：552-7.莫泰吉，A.，K. Kuntz，A. Majeed，S. Smith和K.明哥2006.酿酒酵母中泛素和SUMO连接酶对总染色体重排的调节。分子细胞生物学26：1424-33。Smirnova，M.，和H.L.之间。克莱恩2003.无错误损伤旁路复制后修复途径在维持基因组稳定性中的作用。Mutat Res. 532：117-35.乌尔里希2005. RAD 6通路：泛素和SUMO对DNA损伤旁路和诱变的控制。化学生物化学6：1735-43.