INVOLVEMENT OF C-TERMINAL STRUCTURAL ELEMENTS OF EQUINE INFECTIOUS-ANEMIA VIRUS REVERSE-TRANSCRIPTASE IN DNA-POLYMERASE AND RIBONUCLEASE-H ACTIVITIES

In order to investigate the modes of DNA synthesis supported by the 66 and 51 kDa subunits of equine infectious anemia virus reverse transcr iptase (EIAV RT), recombinant p66 polypeptides containing a modified r ibonuclease H (RNase H) domain were purified and evaluated. Defined he teropolymeric template-primer combinations and high-resolution gel ele ctrophoresis provided a qualitative evaluation of DNA polymerase and R Nase H activities, while DNase I footprinting revealed features of rep lication complexes containing the truncated enzymes. Removal of alpha- helix E ' and the conserved beta 5 '-alpha E ' ''His-loop'' in p66 Del ta 20 RT uncouples the RNase H activities, alters affinity for templat e-primer and dictates how the replicating enzyme responds to secondary structure on both DNA and RNA templates. Despite these alterations, D Nase I footprinting shows no major difference in the overall structure of DNA-directed DNA synthesis complexes. In contrast, removing 47 C-t erminal residues, which includes alpha-helix D ', beta-strand 5 ' and alpha-helix E ', yields an enzyme with distributive DNA polymerase pro perties closely resembling the purified p51 subunit. (C) 1996 Academic Press Limited