The goal of this proposal is to investigate the interaction of a eucaryotic DNA polymerase with its primer-template. Our approach involves photo-crosslinking of oligonucleotide primer- templates to active site peptides of the multi-subunit DNA polymerase. The DNA primase activity of polymerase alpha and the 3' - 5' exonuclease active site of DNA polymerase epsilon will be investigated using modifications of this basic procedure. Two different classes of photoactive nucleotide analogs will be used in this work. In the first, (5-azido- dUTP, 8-azido-dATP and 8-azido-dGTP) the photoactive azido group is attached directly to the pyrimidine or purine base. The primary amino group of this deprotected oligonucleotide will then be reacted with a series of photoactive, heterobifunctional crosslinking reagents to generate oligonucleotide primers with specifically-positioned, cleavable, photoactive nucleotide analogs. The ultimate goal of this proposal is to isolate and identify by sequence analysis, specific active site tryptic peptides crosslinked to oligonucleotide primer-templates. By mapping the active sites of DNA polymerase first to specific subunits of the complex, and later to specific tryptic peptides within the subunit, we hope to begin to understand the structural and functional organization of this remarkable enzyme complex.
