The overall goal of our research remains the investigation of the mechanism by which chromosomes replicate in eukaryotic cells using yeast as a model system. The progress made during the last two years of this project has defined our goals and our research is now centered on (i) multimeric DNA polymerase alpha with associated polymerase, primase, 5'->3'exonuclease, ssDNA dependent ATPase, and DNA helicase activities; (ii) a replication protein A (RP-A) dependent helicase; (iii) an origin binding protein that we have cloned. We have gained insight and isolated some of the proteins and enzymes that are likely involved in the initial stages of DNA replication. With regard to the multimeric DNA polymerase alpha, our goals are: (i) identification and isolation of the ATPase, helicase (helicase A), and 5'->3' exonuclease subunits, and characterization of their enzymatic activities; (ii) structural analysis of the polypeptides and preparation of polyclonal antibodies against each of these subunits; (iii) N-terminal amino acid sequencing, isolation, and characterization of genes for the exonuclease, ATPase, and helicase subunits. We plan to amplify and express the genes for the subunits and activities of multiprotein pol alpha complex with the following goals: (i) PCR amplify the genes for the multiprotein pol alpha subunits and carry out high level expression using E. coli or yeast expression vectors; (ii) structure function analysis of the expressed polypeptides; (iii) analyze the function(s) of the various subunits in DNA synthesis, processivity, and fidelity of the pol alpha complex. We have purified a 48 kDa novel RP-A dependent helicase (helicase B) to homogeneity. Our plan is to characterize the enzymatic activities of these helicases A & B and determine their roles in DNA replication. We have isolated the gene for an origin binding protein (OBP), that is distinct from the more abundant ARS binding factor I. Our present studies indicate that this protein exists as a part of a larger polypeptide complex. We have developed a tentative purification scheme for this complex. Our goals for deciphering the structure and function of this complex are as follows: (i) isolation of OBP complex in native form; (ii) identification and analysis of accessory proteins of OBP, if any; (iii) completion of sequencing and expression of the OBP.
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