The overall goal of this project is to elucidate the structure, function and regulation of the mammalian replication complex. The focus of the work will be DNA polymerase delta, the only mammalian DNA polymerase with intrinsic 3' - 5' exonuclease activity capable of correcting errors of incorporation. The major thrust of this project is to identify, purify and characterize the subunits of DNA polymerase delta holoenzyme and other accessory proteins that are part of this multiprotein complex, which may function as a replication apparatus in mammalian cells. Another objective is to reconstitute active replication complexes from purified components. Several strategies wil be employed, including protein affinity and immunoaffinity chromatography, to identify and purify components of the replication complex. Cloned cDNAs which encode DNA polymerase delta holoenzyme subunits or accessory proteins will also be prepared as additional tools to investigate the structure, function and regulation of the mammalian replication complex. Previous work has resulted in the identification and purification to homogeneity of an auxillary protein for DNA polymerase delta which enables the core enzyme to utilize template/primers containing long stretches of single stranded template. Thus it is now possible to investigate the effects of putative replication complex components on the efficiency, processivity and fidelity with which DNA polymerase delta replicates primed natural DNA templates.
