Enzymes that catalyze the replication of DNA first bind to a nucleic acid complex and then, in general, add many nucleotides to the growing chain before dissociating from the nucleic acid. It is this movement or """"""""translocation"""""""" of the enzyme along the nucleic acid chain, and its relationship to the other steps of the polymerization reaction, that is the subject of this investigation. Rapid rate measurements will be used to resolve the rate of phosphodiester bond formation from the rate of translocation in the reaction catalyzed by DNA polymerase I from E. coli. Complementary oligonucleotides will be synthesized with defined base sequences and used to determine which nucleotide residues of the primer and template chains are in polymerase binding sites at the various stages of the polymerization reaction. From these studies we hope to obtain a dynamic molecular picture of how the enzyme translocates along the nucleic acid chain. The regulation of DNA replication is in part due to the processivity of the DNA polymerases. In the cell this process is controlled by a multitide of priming and binding proteins. However the capacity to copy a nucleotide chain without dissociation exists in systems that contain only the polymerase and the nucleic acid. Thus an understanding of the simplified system will aid our understanding of cellular replication. In addition, knowledge of the molecular details of the polymerase-nucleic acid interaction is essential if rational attempts are to be made to design compounds to control DNA replication.
