HIV-1 replication is inhibited by the incorporation of chain-terminating nucleotides at the 3 end of the growing DNA chain. Here we show a nucleotide-dependent reaction catalyzed by HIV-1 reverse transcriptase that can efficiently remove the chain-terminating residue, yielding an extendible primer terminus. Radioactively labeled 3-terminal residue from the primer can be transferred into a product that is resistant to calf intestinal alkaline phosphatase and sensitive to cleavage by snake venom phosphodiesterase. The products formed from different nucleotide substrates have unique electrophoretic migrations and have been identified as dinucleoside tri-or tetraphosphates. The reaction is inhibited by dNTPs that are complementary to the next position on the template (K i Ϸ 5 M), suggesting competition between dinucleoside polyphosphate synthesis and DNA polymerization. Dinucleoside polyphosphate synthesis was inhibited by an HIV-1 specific non-nucleoside inhibitor and was absent in mutant HIV-1 reverse transcriptase deficient in polymerase activity, indicating that this activity requires a functional polymerase active site. We suggest that dinucleoside polyphosphate synthesis occurs by transfer of the 3 nucleotide from the primer to the pyrophosphate moiety in the nucleoside dior triphosphate substrate through a mechanism analogous to pyrophosphorolysis. Unlike pyrophosphorolysis, however, the reaction is nucleotide-dependent, is resistant to pyrophosphatase, and produces dinucleoside polyphosphates. Because it occurs at physiological concentrations of ribonucleoside triphosphates, this reaction may determine the in vivo activity of many nucleoside antiretroviral drugs.

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Lambda exonuclease is a highly processive 5'-->3' exonuclease that degrades double-stranded (ds)DNA. The single-stranded DNA produced by lambda exonuclease is utilized by homologous pairing proteins to carry out homologous recombination. The extensive studies of lambda biology, lambda exonuclease enzymology and the availability of the X-ray crystallographic structure of lambda exonuclease make it a suitable model to dissect the mechanisms of processivity. lambda Exonuclease is a toroidal homotrimeric molecule and this quaternary structure is a recurring theme in proteins engaged in processive reactions in nucleic acid metabolism. We have identified residues in lambda exonuclease involved in recognizing the 5'-phosphate at the ends of broken dsDNA. The preference of lambda exonuclease for a phosphate moiety at 5' dsDNA ends has been established in previous studies; our results indicate that the low activity in the absence of the 5'-phosphate is due to the formation of inert enzyme-substrate complexes. By examining a lambda exonuclease mutant impaired in 5'-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of lambda exonuclease has been elucidated. We propose a model in which processivity of lambda exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation.

Removal of 2,3-didehydro-3-deoxythymidine-5-monophosphate (d4TMP) from a blocked DNA chain can occur through transfer of the chain-terminating residue to a nucleotide acceptor by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). ATP-dependent removal of either d4TMP or 3-azido-3-deoxythymidine-5-monophosphate (AZTMP) is increased in AZT resistant HIV-1 RT (containing D67N/K70R/ T215F/K219Q mutations). Removal of d4TMP is strongly inhibited by the next complementary deoxynucleoside triphosphate (50% inhibitory concentration [IC 50 ] of ϳ0.5 M), whereas removal of AZTMP is much less sensitive to this inhibition (IC 50 of >100 M). This could explain the lack of cross-resistance by AZT-resistant HIV-1 to d4T in phenotypic drug susceptibility assays.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and other retroviral RTs lack 3Ј-5Ј exonuclease activity (2, 30) but can remove 3Ј-terminal chain-terminating residues from blocked DNA chains through a nucleotide-dependent mechanism leading to production of dinucleoside polyphosphates (23, 24) or through pyrophosphorolysis (the reversal of polymerization) (1,4,13,29). We have recently shown (23) that HIV-1 RT containing the 3Ј-azido-3Ј-deoxythymidine (AZT) resistance mutations D67N, K70R, T215F, and K219Q (67/70/215/219 mutant RT) removes AZT-5Ј-monophosphate (AZTMP) from blocked primer-templates through the nucleotide-dependent mechanism more efficiently than does wildtype (WT) RT. The mutant enzyme also removes 2Ј,3Ј-dideoxyadenosine-5Ј-monophosphate (ddAMP) from blocked DNA chains more efficiently than does WT RT. Removal of ddAMP is strongly suppressed by physiological concentrations of deoxynucleoside triphosphates (dNTPs), whereas removal of AZTMP is much less sensitive to this inhibition (23).The chain terminator 2Ј,3Ј-didehydro-3Ј-deoxythymidine-5Ј-triphosphate (d4TTP) is efficiently incorporated into growing DNA chains by HIV-1 RT (39). Resistance to d4T can arise in cell culture through a valine-to-threonine mutation at position 75 (19, 21, 32); however, this mutation is rarely observed in HIV-1 from d4T-treated individuals (6,9,17,22,27,35). Instead, mutations associated with AZT resistance, including M41L, D67N, K70R, L210W, and T215Y/F, are frequently selected (6,8,21,22,27,32,35). The selection of AZT resistance mutations by d4T in the absence of AZT is unexpected, since phenotypic assays show little, if any, cross-resistance between these drugs (20,22). Nonetheless, clinical studies have shown that prior exposure to AZT reduces the efficacy of subsequent treatment with d4T (17), and the presence of AZT resistance mutations is correlated with reduced suppression of viral load in response to d4T-containing therapies (15, 25). These results suggest that the phenotypic assays do not fully reflect the in vivo sensitivity of HIV-1 replication to d4T.In an effort to understand the biochemical basis for the lack of cross-resistance by AZT-resistant mutants to d4T, we have investigated the ability of WT and 67/70/215/219 m...

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.