Analysis of Spt16-Pob3 in Promoting Replication and Transcription in Yeast

University Of Utah, Salt Lake City UT

Investigators

Abstract

Formosa The Spt16 and Pob3 proteins from the yeast Saccharomyces cerevisiae form a stable, abundant, nuclear heterodimer. Homologs of these proteins have been found in all eukaryotes tested, and have been purified as stable heterodimers from human and frog sources. One of these conserved heterodimers, the human FACT complex, enhances the ability of RNA polymerase II to progress past template sites associated with nucleosomes, suggesting that Spt16-Pob3 is a chromatin-specific transcription elongation factor. Another homolog, the frog DUF complex, is needed for normal DNA replication in oocyte extracts, suggesting that Spt16-Pob3 is a DNA replication factor. Data from the yeast system support both of these models, and point to chromatin as the common point of action. Mutations in SPT16 lead to global changes in transcription similar to the changes caused by some histone mutations, and display interactions with putative chromatin factors. The Spt16-Pob3 dimer binds to DNA Polymerase a, and interacts genetically with several replication proteins. While the role of chromatin in modulating transcription has been widely studied, neither the mechanism by which Spt16-Pob3 affects chromatin nor the role played by chromatin in DNA replication have been firmly established. The goal of this research is to further analyze the activity of the Spt16 and Pob3 proteins in DNA replication as well as in transcription. Yeasts with mutations in SPT16 and POB3 will be studied to determine whether DNA replication is aberrant by examining replication intermediates directly and by testing genetically for interactions with replication factors and DNA damage checkpoints. Localization of Spt16-Pob3 to promoters and replication origins will be tested to see if this varies in an informative way with the activation of these sites. The progression of replication forks in the mutants will be measured to assess whether Spt16-Pob3 contributes to the ability of DNA polymerases to act on chromatin in vivo as FACT does with RNA polymerase in vitro. These experiments will allow dissection of models concerning the function of Spt16-Pob3 in both replication and transcription. Other factors that act with Spt16-Pob3 will be sought using both genetic and physical methods. High copy suppressors and extragenic enhancer mutants will be identified in collaboration with a set of High School students. This will provide information regarding factors that interact with Spt16-Pob3, and will also provide a non-trivial research experience for a broad group of students. Affinity chromatography will also be performed with purified Spt16-Pob3 to identify physical interactions. DNA molecules contain instructions for building cells. Using this information requires making temporary working copies of the instructions as RNA molecules in a process called transcription. DNA copies of the cell's DNA must be also be made periodically so that each daughter cell can have a set of the instructions. This is called replication. While very different in detail, transcription and replication both act on the cell's DNA molecules and so have many common needs. For example, the DNA inside of cells is bound tightly by proteins to form a complex called chromatin. The enzymes that must copy the DNA, either into RNA or DNA, must at least temporarily disturb the binding of the chromatin proteins. While this process has been partially studied for transcription, it has not been well-characterized for replication and might use many of the same factors. The research supported by this grant has identified two proteins, called Spt16 and Pob3, that are found in all eukaryotes tested. These proteins appear to act together to assist both replication and transcription enzymes in the process of untangling DNA from chromatin during the copying processes. The project will now focus on learning how these proteins work. Cells with damaged versions of the two proteins will be examined to see if transcription and replication occur normally. Additional genetic changes that either make these mutated cells function more normally, or that make them less capable of maintaining themselves, will be sought to provide clues about the function of the factors. This will be done along with a group of High School students to help them learn not only about transcription and replication factors, but about how science is a process used to find answers to questions. This research therefore addresses questions about the fundamental mechanisms of replication and transcription, and also provides an educational experience to a broader group.