RAD5 INTERACTING PROTEIN SEARCH BY YEAST TWO HYBRID SCREENING

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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.