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。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。持续停滞的复制叉塌陷并引起基因组不稳定性（Kolodner等，2002）。在酵母中，通过绕过translesy合成聚合酶绕过DNA损伤，或通过将模板切换到姐妹染色单体的新生链（Smirnova and Klein，2003; Ulrich，2005）来解决停滞的复制叉。通过修饰增殖细胞核抗原（PCNA）来调节这两种途径的决定。 PCNA是由RAD18单泛素化的，并在单液液化的PCNA上由Rad5进行了进一步的聚泛素化（Ulrich，2005）。我们发现RAD18和RAD5通过PCNA的多泛素化抑制了染色体重排（GCR）（Motegi等，2006）。该抑制机制与细胞周期检查点途径有关。在这项研究中，我们还证明了另一个小型泛素样修饰剂（SUMO）连接酶SIZ1通过修饰PCNA通过与Helicase SRS2和Rad51依赖性重组机械的相互作用而促进GCR形成，从而拮抗GCR形成。我们的数据将RAD5和RAD18放在细胞周期检查点依赖性GCR抑制途径中，并添加了SIZ1-SRS2定向RAD51依赖性重组途径作为第三个GCR增强途径。为了研究RAD5途径如何通过模板开关来指导DNA损伤途径，我们与Marrissa Vignali（U. Washington）建立了合作，以通过酵母两种杂交筛选来找到酵母Rad5相互作用。我们将在正常和DNA损伤条件下筛选整个酵母打开的阅读框。从这些筛选中鉴定出的蛋白质编码蛋白质的基因将被测试是否功能抑制GCR形成。 参考）Kolodner，R.D.，C.D。 Putnam和K. Myung。 2002。维持酿酒酵母中的基因组稳定性。科学。 297：552-7。 Motegi，A.，K。Kuntz，A。Majeed，S。Smith和K. Myung。 2006。在酿酒酵母中，泛素和相扑酶调节总染色体重排。摩尔细胞生物。 26：1424-33。 Smirnova，M。和H.L. Klein。 2003。无误损伤旁路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（H.D.） 2005。RAD6途径：泛素和SUMO对DNA损伤旁路和诱变的控制。 Chembiochem。 6：1735-43。