An expanded model of replicating human immunodeficiency virus reverse transcriptase.
Replication complexes containing wild-type and RNase H-deficient p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) were analyzed by DNase I and S1 footprinting. While crystallography and chemical footprinting data demonstrate that 15-18 bases of primer and template occupy the DNA polymerase and RNase H active centers, enzymatic footprinting suggests that a larger portion of substrate is encompassed by the replicating enzyme. Independent of the position of DNA synthesis arrest, template nucleotides +7 to -23 and primer nucleotides -1 to -25 are nuclease resistant. On both DNA strands, position -20 remains accessible to DNase I cleavage, suggestive of an alteration in nucleic acid structure between exiting the RNase H catalytic center and leaving the C-terminal p66 domain. A model of HIV-1 RT containing an extended single-stranded template and duplex region was constructed on the basis of the structure of an RT/DNA complex. Mapping of footprint data onto this model shows consistency between biochemical and structural data, implicating a contribution from domains proximal to the catalytic centers.
Wöhrl
B M
BM
Division of Infectious Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Tantillo
C
C
Arnold
E
E
Le Grice
S F
SF
D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003850:Deoxyribonuclease I; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D003850:Deoxyribonuclease I; D016914:Ribonuclease H