MODULATION OF HIV-1 REVERSE TRANSCRIPTASE AND FAMILY A DNA POLYMERASE PRIMER-TEMPLATE BINDING

Abstract

Polymerases are enzymes used by all cellular and viral organisms to replicate their genomes. The human immunodeficiency virus (HIV) polymerase, reverse transcriptase (RT), uses a single-stranded RNA template to create double-stranded DNA during the course of the viral life cycle. Successful reverse transcription relies on the speed of catalysis and the ability of the enzyme to stay bound to the template during synthesis. I demonstrate that both of these properties can be modulated by the presence of different divalent cations, fundamentally altering the behavior of HIV RT. In the presence of 2 mM Mg<super>2+</super>, the HIV RT primer-template complex has a half-life of 1.7&plusmn;1.0 min, incorporating nucleotides at a maximum rate of 3.5 nucleotides (nt) per second (average speed 1.4&plusmn;0.4 nt/sec). Substituting 2 mM Mg<super>2+</super> with 400 &#956;M Zn<super>2+</super> dramatically slows the speed of catalysis (maximum 0.1 nt/sec, average 0.022&plusmn;0.003 nt/sec) and promotes primer-template complexes that last hours (half-life of 220&plusmn;60 min). These profound changes to the enzyme's function critically inhibit reverse transcription, even in the presence of optimal concentrations of Mg<super>2+</super>. In addition to the cation composition during reverse transcription, previous studies have demonstrated that the sequence of the primer-template substrate can also affect the duration of a RT-primer-template complex. In light of this discovery, I investigated the tendency of two Family A DNA polymerases, the <i>Thermus aquaticus</i> DNA polymerase (Taq pol) and the Klenow fragment from <i>Escherichia coli</i> DNA polymerase I (Klenow), to selectively and tightly bind primer-template complexes. Using Primer-Template Systematic Evolution of Ligands by Exponential Enrichment (PT SELEX), I determined that both <i>Taq</i> pol and Klenow tightly bind to sequences containing regions that match the initiation and melting domains of promoters for the structurally similar bacteriophage T7-like RNA polymerases. This suggests a shared sequence preference that might be present in all Family A DNA polymerases, derived from a common ancestor. I plan to exploit this primer-template binding preference to advance biotechnologies utilizing these enzymes.