9513248 McHenry The DNA polymerase III holoenzyme is the replicative complex of E. coli, responsible for the synthesis of the majority of the chromosome. The replicative complexes of all cellular systems are closely related. They all consist of a special replicative polymerase, structurally identical sliding clamps with which the polymerase associates and a clamp setting apparatus. In E. coli, these components correspond to the DNA polymerase III ((((), the ( "sliding clamp", and the 5-protein DnaX clamp setting apparatus. The holoenzyme contains 10 different subunits, all available in 100 mg quantities from overproducing strains. 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. Using photoreactive nucleotides placed at unique positions within long primers, my laboratory has located the ( catalytic subunit (3'-nucleotides 1-13), the ( (DnaX)-complex (contact at nucleotide 18) and the processivity factor (nucleotide 22) within the initiation complex. In this proposal, experiments are outlined to extend this analysis in order to identify subunit contacts with the template, particularly in front of the primer and regions within the template with short primers of ca. 10 nucleotides such as those used to prime DNA replication. Placement of photoreactive nucleotides at specific locations in templates of designed sequence will permit the replicative complex to be "walked" into specific positions by the addition of a limited number of dNTPs. This will permit changes in the composition and the contacts within the replicative complex to be determined as it progresses from initiation to elongation. The positioning of subunits relative to others within the replicative complex will be determined by chem ical protein-protein cross-linking. Initial focus will be on the "zero-length" cross-linker EDC. These carbodiimide-induced intersubunit cross-links will be analyzed at the sequence level to permit regions of subunit-subunit contact to be localized. Fluorescence energy transfer will be used to determine the distance between subunits. Primary focus will be on the distances between subunits that do not cross-link to one another. Concurrent determination of the distances of these subunits to the primer terminus and the ( subunit will permit triangulation and placement of the studied components within the replicative complex. These experiments are supported by our demonstrated ability to locate the ( subunit 65 A from the 3'-antepenultimate nucleotide in primers, a position verified by photocross-linking. Additionally, pilot experiments will be conducted collaboratively using electron microscopy to examine the structure of the replicative complex. Initial focus will be upon a direct visual test of the dimeric polymerase hypothesis. This proposed work will yield the first detailed structure of a prototypical replicative complex at the level of subunit organization. %%% The 10-protein apparatus that duplicates chromosomes prior to cell division is closely conserved between bacterial and higher cells. The purpose of this proposed study is to determine the physical arrangement and the contacts of the components of a prototypical DNA replicative machine. This will be accomplished by (i) placing analogs of DNA bases at specific points in a model chromosome that chemically attach to proteins when flashed with light to determine the linear arrangement of the replication proteins relative to DNA both in a static and an active elongation mode; (ii) direct chemical protein-protein cross-linking to determine which proteins touch one another, (iii) fluorescence energy transfer, a technique that uses a spectroscopic (light) ruler to determine the distances between the components that do not contact one another and (iv) electron microscopy to directly visualize relative positions of proteins and DNA. These complementary methods will permit a description of the geometry of a prototypical replicative complex and permit the design of specific experiments to test the function of protein subunits implied by their position(s) within the complex. ***
