Enzyme-nucleic acid interactions represent a fundamental area of research that requires structural and functional data in order to elucidate general principles. Although there are a large number of enzymes which catalyze reactions for nucleic acid substrates, only twelve enzyme-nucleic acid complex structures have been reported to date. This proposal describes structural and functional studies designed to address the importance of enzyme-nucleic acid interactions in polymerization by reverse transcriptase (RT). The relative simplicity of RTs, which retain effective replicative capacity but lack the associated components of other replicative polymerases, make them ideal for studying the process of polymerization. RTs couple two catalytic activities, an RNA- or DNA-directed DNA polymerase activity and a ribonuclease H (RNaseH) activity, in order to make a double-stranded DNA copy of the single-stranded RNA genome of the retrovirus. The retroviral DNA is then integrated into the host genome by the retroviral integrase, and the host's cellular machinery is utilized to produce more retrovirus. The epidemic outbreak of AIDS caused by human immunodeficiency virus type l (HIV- l) has focused a great deal of research efforts on HIV- 1 RT as a target for drug therapy. We propose to use x-ray crystallographic methods to determine the structures of a nucleic acid complex with a catalytic fragment of Moloney murine leukemia virus (MMLV) RT and- complex with the full- length MMLV RT. Our apprdach is to compare conserved features of the structures of MMLV RT and HIV- l RT in order to understand how the enzyme works. Basic and detailed knowledge of catalysis and substrate interactions in RT wlll further efforts in the development of effective inhibitors. From our analyses of the MMLV RT-nucleic acid complex structures, we propose to elucidate a detailed mechanism of catalysis for polymerization by RT, to determine important interactions with nucleic acid, and to identify the structural basis of substrate specificity. In addition, we plan to pursue site-directed mutagenesis studies in which specific amino acid residues will be substituted based on our structural analyses, and functional properties of the mutant RTs will be characterized. Crystal structures will be determined for interesting mutant RTs complexed with nucleic acid and comparisons made to the wild-type enzyme complex. We expect that insights derived from these studies will extend to other related polymerases and our general knowledge of enzyme-nucleic acid interactions.
