RAD5 INTERACTING PROTEIN SEARCH BY YEAST TWO HYBRID SCREENING

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。持续停滞的复制分叉崩溃并导致基因组不稳定(Kolodner等人，2002年)。在酵母中，停滞不前的复制分叉可以通过跨损伤合成聚合酶绕过DNA损伤或通过模板切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对增殖细胞核抗原的修饰来调节的。增殖细胞核抗原是由Rad18单素化的，并进一步由Rad5在单元化的增殖细胞核抗原上进行多泛素化(Ulrich，2005)。我们发现Rad18和Rad5通过增殖细胞核抗原的多泛素化抑制总染色体重排(GCR)(Motegi等人，2006年)。这种抑制机制与细胞周期检查点通路有关。在这项研究中，我们还证明了另一种小的泛素样修饰物(SUMO)连接酶Siz1通过与解旋酶Srs2和依赖RAD51的重组机制相互作用来修饰增殖细胞核抗原，促进GCR的形成，从而发挥拮抗作用。我们的数据将Rad5和Rad18置于细胞周期检查点依赖的GCR抑制通路中，并添加了Siz1-Srs2导向的RAD51依赖的重组通路作为第三个GCR增强通路。为了研究Rad5途径如何通过模板切换引导DNA损伤避免途径的机制，我们与华盛顿大学的Marrissa Vignali合作，通过酵母双杂交筛选寻找酵母Rad5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。从这些筛选中识别出的编码蛋白质的基因将被测试它们是否具有抑制GCR形成的功能。参考文献：Kolodner，R.D.，C.D.Putnam，K.Myung。2002年。酿酒酵母基因组稳定性的维持。科学。297：552-7。莫特吉，A.，K.昆茨，A.Majeed，S.Smith和K.Myung。2006年。泛素和相扑连接酶对酿酒酵母染色体总重排的调控。Mol Cell Biol.26：1424-33。Smirnova，M.和H.L.Klein。2003年。无错损伤旁路复制后修复通路在维持基因组稳定性中的作用。穆塔特书532：117-35。乌尔里希，H.D.，2005。RAD6途径：泛素和相扑对DNA损伤的控制、旁路和突变。化学生物化学。6：1735-43。

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.