The DNA polymerase Ill holoenzyme is the replicative complex of E. coli, responsible for the synthesis of the majority of the chromosome. The holoenzyme contains at least 10 different subunits, most present in two or more copies, forming a complex of approximately 800,000 daltons. This replicative complex exhibits many properties that distinguish it from simpler polymerases. These properties include a high rate of elongation, the ability to form an ATP-dependent highly processive clamp on the DNA template, and the ability to function as an asymmetric dimer with distinguishable leading and lagging strand polymerases. Our knowledge of the function of this enzyme surpasses our knowledge of its structure. We need to know the subunit arrangements of this enzyme and its points of contact with the template-primer so that more sophisticated testable hypotheses can be formulated regarding the function of the individual components. We intend to take a tripartite approach to establishing the structure of the DNA polymerase III holoenzyme at the level of subunit arrangement. The points of subunit-template and subunit-primer contact will be identified by a photo-affinity labeling approach. We will make a series of'template-primers each containing a photo-affinity label attached to a unique nucleotide. Identification of which subunits cross-link to each template-primer will identify a contact and will provide orientation points upon which we can build up a holoenzyme structure. (ii) We will identify subunit contacts by chemical cross-linking. This will permit us to assemble a group of testable structures for holoenzyme. (iii) Fluorescence energy transfer will be used to determine distances between the template-primer, active sites and individual subunits. This will provide checks of the results obtained by the techniques described above and will provide information about the spatial relationship of subunits not obtained by other methods. We also intend to address structural issues regarding the asymmetric dimer hypothesis for the DNA polymerase III holoenzyme.
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