Alterations to the primer grip of p66 HIV-1 reverse transcriptase and their consequences for template-primer utilization.
Alanine scanning mutagenesis was undertaken to evaluate the structural significance of Met230-His235 of the 66 kDa subunit of p66/p51 human immunodeficiency virus reverse transcriptase (HIV-1 RT). Together with Glu224-Trp229, these residues provide the framework of the p66 "primer grip", whose proposed role is maintaining the primer terminus in an orientation appropriate for nucleophilic attack on an incoming dNTP. Of these residues, altering Leu234 results in a p66 subunit incapable of associating into heterodimer. The remaining selectively mutated enzymes were successfully reconstituted and purified to homogeneity for evaluation of RT-associated activities. We show here that alterations to any residue within the p66-Trp229-Met230-Gly231-Tyr232-quartet alter functions associated with both the DNA polymerase and ribonuclease H (RNase H) domains. Detailed analysis of mutant p66Y232A/p51 with an intact or a model "precleaved" RNA-DNA hybrid suggests an altered RNase H phenotype could result from relocation of template-primer in the nucleic acid binding cleft. As a consequence, template nucleotide-8 is positioned in the immediate vicinity of the RNase H catalytic center rather than nucleotide-17.
Ghosh
M
M
Center for AIDS Research and Division of Infectious Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Jacques
P S
PS
Rodgers
D W
DW
Ottman
M
M
Darlix
J L
JL
Le Grice
S F
SF
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D011995:Recombination, Genetic; D016914:Ribonuclease H / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H