ENZYMATIC MECHANISMS OF DNA SYNTHESIS

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The goal of this work is to understand the mechanisms by which DNA polymerases replicate chromosomal DNA. Polymerases required for this process are multisubunit complexes of proteins. Two such enzyme complexes are under investigation, E. coli DNA polymerase III holoenzyme and calf DNA alpha polymerase. The experimental approach is to determine the functional contribution of each subunit in the complexes during DNA synthesis in vitro. Purified preparations of DNA polymerase III holoenzyme have been obtained which consist of at least seven different protein subunits. Additionally, three stable subassemblies of DNA polymerase III holoenzyme have also been isolated which lack one or more of the protein subunits found in the holoenzyme. In each of these subassemblies of DNA polymerase III holoenzyme a correlation exists between the absence of individual subunits and the loss of one or more properties associated with the holoenzyme. These properties include ATP dependent formation of stable initiation complexes with DNA, ability to use DNA coated with polyamines and binding proteins for highly processive synthesis, and proper dissociation at natural synthesis termination sites. We are examining the effects of the presence of particular combinations of subunits on these properties, and in this way hope to identify the functional role of each subunit. Recent evidence suggests that the holoenzyme may be a functional dimer in vivo. We are using electron microscopy to detect interaction of one polymerase molecule with two substrates, and measure synthesis on each substrate. The calf DNA alpha polymerase is similarly intricate and also has been difficult to purify. However, we have recently developed a method for immunoaffinity chromatography purification of a high molecular weight form of this enzyme. Experiments are proposed to characterize the subunit structure of the enzyme, and to establish functional properties of individual subunits. The effects of ATP, calf DNA binding protein UP1 and calf DNA dependent ATPases on the synthetic properties of this polymerase will be determined. It is expected that by this method the combination of polymerase subunits and replication associated proteins which operate in vivo can be assembled in vitro.