Nucleotide-Induced Stable Complex Formation by HIV-1 Reverse Transcriptase

• Published in: Biochemistry (Easton) 1997-05, Vol.36 (19), p.5749-5757
• Main Authors: Tong, Wenjing, Lu, Chang-De, Sharma, Satish K, Matsuura, Suzanne, So, Antero G, Scott, Walter A
• Format: Article
• Language: English
• Subjects: AIDS/HIV; Deoxyadenine Nucleotides - metabolism; DNA Primers; Electrophoresis, Polyacrylamide Gel; HIV Reverse Transcriptase - genetics; HIV Reverse Transcriptase - metabolism; human immunodeficiency virus 1; Nucleic Acid Conformation - drug effects; Templates, Genetic; Thionucleotides - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi962410z

Nondenaturing gel electrophoresis was used to study the nucleotide substrate-induced conformational change in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Dead-end complex was formed between HIV-1 RT, dideoxynucleotide chain-terminated primer, and DNA template in the presence of deoxynucleotide triphosphate (dNTP) complementary to the next position on the template. Complexes which form in the absence of the next complementary dNTP were disrupted by adding excess poly(rA)/oligo(dT) or heparin just prior to electrophoresis. Dead-end complex formation by noncomplementary dNTP's or ribonucleotides was at least 2000-fold less efficient than with the complementary nucleotide. When dA was the next nucleotide on the template, analogues of dTTP supported dead-end complex formation with increased apparent K d (dTTP < dideoxy-TTP ≈ α-thio-dTTP < dUTP < 3‘-azidothymidine triphosphate). A similar relationship was observed for dGTP analogues across from dC on the template (dGTP < dideoxy-GTP < α-thio-dGTP