CHARACTERIZATION OF ESSENTIAL PROTEINS INVOLVED IN CHROMOSOME SEGREGATION

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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. Normal cell division requires that all the chromosomes are duplicated and separated equally into the two daughter cells. During mitosis, sister chromatids are held together by protein complexes known as cohesins (by a process known as sister chromatid cohesion), until the metaphase to anaphase transition. Defective cohesion can lead to aneuploid cells by an increased rate of gain or loss of chromosomes (known as chromosome instability (CIN)). In mammalian systems, CIN may contribute to cancer progression and is exhibited by a variety of cancers, yet the mechanism by which it occurs is poorly understood. The budding yeast Saccharomyces cerevisiae has been useful for identifying genes that are involved in maintenance of genomic integrity and conserved in mammals. A recent genetic screen of a Ts mutant resource in the Hieter laboratory yielded three novel essential genes that lead to CIN, cohesion defects and sensitivity to DNA damaging agents when mutated. In order to fully understand the biological function of the corresponding proteins, a primary goal of this project is to identify interacting proteins that may yield information about which specific biological process these proteins are involved in. The primary technique used will be tandem affinity purification (TAP) in order to isolate the proteins of interest as part of intact complexes and to identify interacting partners. The TAP method has been shown to be useful for isolating biologically active complexes of sufficient purity for mass spectrometric analysis and protein identification. The results obtained from TAP will not only expand our knowledge of protein interaction networks in yeast but will also provide a starting point to further characterize the role of these novel proteins in DNA repair and sister chromatid cohesion establishment and/or maintenance.