Biochemical & Genetic Analysis of Yeast SPN

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DESCRIPTION (provided by applicant): The yeast Saccharomyces cerevisiae encodes two proteins, Sptl6 and Pob3, that are each highly conserved among eukaryotes, including humans. Sptl6 and Pob3 function as a heterodimer, and this factor is required for both DNA replication and RNA transcription since cells lacking normal proteins display errors in both processes. Sptl6-Pob3 influences both how often transcripts are made and the precise site chosen for initiation. It also promotes elongation of transcripts. The surprising breadth of the roles of this factor can be explained by a single simple activity: the ability to modulate the properties of nucleosomes. Since these structures affect all regions of the eukaryotic genome, they are involved in all processes that involve the genome, from establishing initiation sites for transcription and replication, to the progression of DNA and RNA polymerases, to the packaging and segregation of genomic copies. Sptl6-Pob3 is unlike its homologs from other eukaryotes in that it lacks a DNA-binding motif. Physical and genetic methods indicate that it functions together with a DNA-binding protein called Nhp6. Purified Spt 1 6-Pob3 and Nhp6 alter the structure of nucleosomes in vitro in a way that changes their electrophoretc mobility and alters the presentation of the DNA in the nucleosome. Experiments in this proposal explore the nature of the changes induced in nucleosomes by Sptl6-Pob3-Nhp6 (SPN), with the goal of understanding how SPN changes the structure of this fundamental unit of genomic packaging. The function of SPN in cells is then addressed by using genetic methods to test specific models describing steps in replication and transcription that might be promoted by SPN. The effect of diminishing SPN activity is then examined in assays that reveal the formation of transcription and replication initiation complexes in SPN mutants, and the structure of intermediates formed during elongation. Sptl6-Pob3 does not reposition nucleosomes like a standard chromatin remodeling factor, but appears to be a new type of factor that reorganizes nucleosomes. These studies will elucidate activity of this highly conserved factor in modulating the effects of a fundamental component of chromatin, and will indicate how this activity participates in two basic processes of nucleic acid metabolism: transcription and replication.