This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The ultimate goal of our project is developing biochemical and mass spectrometric methods that can reveal stable interactions of proteins with any chosen region of the S. cerevisiae chromosome. Our strategy is to develop a novel method to investigate protein complexes that bind to a given region of DNA, in which we affinity-purify the DNA sequence of interest from a yeast cell lysate. We have chosen to use the enrichment of 2 ?m plasmids from S. cerevisiae as a model case. Two micron plasmids are endogenous circular 6.3 kbp minichromosomes, which are present in most common strains of S. cerevisiae at a copy number of approximately 60 per cell. The plasmid contains one origin of replication and is replicated once, and only once, per cell cycle by the cellular replication machinery. Therefore, it serves as an appropriate model for studying (initiation of) DNA replication. We are optimizing affinity-purification procedures of a specific DNA sequence by enrichment of 2 ?m plasmids by screening for proteins that are involved in DNA replication initiation. For this purpose, 2 ?m plasmids have been modified by insertion of an array of lac operator sequence repeats. A S. cerevisiae strain carrying an expression cassette for green fluorescent protein (GFP) ? lac repressor fusion protein was transformed with a plasmid containing the lac operator repeat sequence and the 2 ?m plasmid origin of replication (pSV1 plasmid). Fluorescence microscopy experiments showed clear fluorescent dots in the cells and suggest that the GFP-lac protein is specifically localized, probably to the lac operator repeats on the pSV1 plasmids. In order to pull-out plasmid circles from cell lysates by affinity-purification, highly specific ?-GFP antibodies conjugated to magnetic Dynabeads are used. After extensive washing of the beads, the plasmids are eluted and the proteins are subsequently analyzed by 1D SDS-PAGE and identified by mass spectrometry. Typical protein patterns show GFP-lac repressor protein, histones in the low molecular weight range and several bands in the high molecular weight range. Analysis of these high-molecular weight proteins is currently performed. Of those, one has been identified as being a putative transcription factor protein, which may play a role in plasmid DNA replication. Ultimately, in principle, any specific sequence in the entire yeast genome can be tagged with an array of lac operon repeats, cleaved off by specific restriction enzymes and affinity-purified as described.
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