A very convenient system has been developed to study the interaction of a DNA polymerase with nucleic acid substrates. The structure of the enzyme reverse transcriptase of HIV-1 complexed with a 19/18-mer template-primer was solved and represents the first image of a DNA polymerase interacting with a template-primer substrate at the polymerase active site.
The aim of the studies proposed here is to engineer a modification on the surface of HIV-1 RT in order to increase its processivity during DNA synthesis. We plan to introduce a 30 amino acid peptide at a specific site on RT utilizing standard molecular biology techniques. The peptide to be engineered corresponds to a structural element present in a highly processive nucleic acid polymerase, the T7 RNA polymerase, not found in RT, a poorly processive enzyme. This structural element is thought to help the polymerase to hold on to the nucleic acid substrate, increasing its processivity. Molecular modeling experiments have been performed in order to identify a suitable for the modification on HIV-1 RT. The processive RT obtained from these studies will be overexpressed and crystallized in the presence of template-primer substrate, and its three- dimensional structure determined by X-ray crystallography. Since only a small portion of the enzyme will be different from that of the wild-type, the structure determination can be accomplished utilizing the straightforward difference Fourier method. Our structural knowledge of processive DNA synthesis is limited. The three-dimensional structure of the Beta-subunit of E. coli DNA polymerase II holoenzyme represents the first insight into this process. However, this subunit is an accessory protein that confers complete processivity onto the polymerization domain of the holoenzyme, and, therefore is not a DNA polymerase in itself. The studies proposed here contemplate the possibility of engineering processivity into the poorly processive HIV-1 RT. Hence, these studies could make a significant contribution to the understanding of the molecular principles of processive DNA synthesis. In addition, we might be able to suggest the design of specific inhibitors that will reduce or block processive DNA or RNA synthesis, which is crucial during the life cycle of viral pathogens.
