DNA polymerase I and primase are the two major chromosomal DNA replication enzymes in the yeast Saccharomyces cerevisiae. Our immediate goal is to delineate the mechanism of these enzymes in the priming and elongation stages of DNA replication. Thus far, we have purified polymerase-free yeast DNA primase and developed assays for the DNA polymerase holoenzyme of yeast. Partial purification of a high molecular weight ( 500,000Da) DNA pol I holoenzyme has been achieved. We plan to complete the purification of the holoenzyme to homogeneity. We would characterize each of the polypeptides that copurify with holoenzyme through immunological and structural studies. We shall determine various holoenzyme-associated activities, location of these activity sites in the polypeptide assembly, and their function in the overall mechanism of action of the holoenzyme. We will determine the in vitro rate of DNA synthesis, fidelity of DNA synthesis of holoenzyme in vitro and processivity of such synthesis. In order to derive conclusions regarding the structure and function of holoenzyme we would determine the influence of various subunits and cofactors such as single stranded DNA binding protein in its various properties. We plan to study the mechanism of protein-nucleotide and DNA- protein interactions, involved in the mechanism of action of holoenzyme through ultraviolet photocrosslinking. This will likely provide insights in its modes of action and complement our mechanistic studies as outline above. We have initiated isolation of yeast primase gene and we plan to complete primase gene isolation and gain insights into its genetic requirement in vivo.
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