DNA polymerase I (Pol I) of E. coli is an enzyme that plays an important role in the cell both during replication, in the processing of Okazaki fragments, and in the repair of damaged DNA. Now that the three-dimensional structure of an active fragment of Pol I (the Klenow fragment) has been solved, this enzyme is undoubtedly the best model system for the study of the molecular details of replication, and for an investigation of the way in which processive enzyme can coordinate synthesis with movement along a macromolecule. We have been collaborating closely with the x-ray crystallographers in interpreting the Klenow fragment structure, and we plan to continue these studies. We believe that the two domains seen in the Klenow fragment structure correspond to separable polymerase and 3'-5' exonuclease (editing) activities; we shall test this idea by investigating the enzymatic properties of the isolated domains. Using a combination of genetic and biochemical techniques, we have obtained evidence supporting the location of the DNA binding site inferred from model building. We have also tentatively identified the polymerase and 3'-5' exonuclease active sites. To confirm the location of these active sites and to identify critical amino acid residues within them, we shall use site-specific and localized mutagenesis techniques to generate altered forms of Klenow fragment. The mutant enzymes will be purified and characterized in vitro in order to determine the effect of each mutation on substrate binding and catalysis. The polymerase-DNA interaction will be examined biochemically by identifying positions of phosphates (relative to the primer terminus) that contact the protein. Further studies on the enzyme-DNA interaction will examine whether the Klenow fragment interacts differently with its DNA substrate in each of its two enzymatic roles, whether there is a change in conformation on binding DNA, and how the enzyme distinguishes between the different types of nicked and gapped duplexes encountered in vivo. While this project has no direct health-related application, it is clear that an improved understanding of those processes in DNA replication and repair that maintain the integrity of the genome should eventually provide insights into the mechanisms of mutagenesis and thus of carcinogenesis.
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