Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 Kd (dTTP < dideoxy-TTP approximately alpha-thio-dTTP < dUTP < 3'-azidothymidine triphosphate). A similar relationship was observed for dGTP analogues across from dC on the template (dGTP < dideoxy-GTP < alpha-thio-dGTP << dITP < dideoxy-ITP). The optimal length of the primer/template duplex region for dead-end complex formation was between 20 and 32 base pairs. Primer-template with a mismatched primer terminus did not support dead-end complex formation, and primer terminated with 3'-azidothymidine formed dead-end complex with 25-fold elevated apparent Kd. By contrast, dead-end complex formation on primer terminated with dideoxy-IMP base paired with dC on the template was more efficient than on primer terminated with dideoxy-GMP. Implications for the mechanisms of discrimination between nucleotide analogues by HIV-1 RT are discussed.
 ...
PMID:Nucleotide-induced stable complex formation by HIV-1 reverse transcriptase. 915 15

Oxidatively modified deoxynucleotide triphosphates (dN(oxo)TPs) present in nucleotide precursor pools may contribute to retroviral mutagenesis as a result of incorporation and ambiguous base pairing during reverse transcriptase mediated replication. We have examined the incorporation of 5-hydroxy-2'-deoxycytosine triphosphate (5-HO-dCTP) and 2'-deoxyinosine triphosphate (dITP) by HIV-1 reverse transcriptase (HIV-1 RT) on DNA and RNA templates of the same sequence in order to evaluate their mutagenic potential. Significant variations in insertion frequencies at homologous nucleotide positions were observed for each dN(oxo)TP, in general favoring the RNA template. A comparison of steady-state kinetics revealed a 10-fold preference for 5-HO-dCTP incorporation opposite G in RNA. Insertion frequencies for dITP were 2- to 20-fold greater on RNA for every base position examined. One exception to this general trend was observed for the insertion of 5-HO-dCTP by HIV-1 RT opposite A, which favored the DNA template by 4-fold. Deoxyinosine triphosphate was inserted opposite C with an 8-fold higher frequency compared to dGTP in RNA, while on DNA templates, the incorporation frequencies were equivalent. However, incorporation of dITP opposite other bases was characterized by relatively low frequencies. The RNA template bias observed for dN(oxo)TP incorporation is discussed in terms of recent efforts to utilize 5-OH-dCTP as an anti-HIV agent.
 ...
PMID:Incorporation of oxidatively modified 2'-deoxynucleotide triphosphates by HIV-1 RT on RNA and DNA templates. 1201 86