Dideoxythymidine Triphosphate Research Articles

HIV Reverse Transcriptase Pre-Steady-State Kinetic Analysis of Chain Terminators and Translocation Inhibitors Reveals Interactions between Magnesium and Nucleotide 3'-OH.

Deoxythymidine triphosphate analogues with various 3′ substituents in the sugar ring (−OH (dTTP)), −H, −N3, −NH2, −F, −O–CH3, no group (2′,3′-didehydro-2′,3′-dideoxythymidine triphosphate (d4TTP)), and those retaining the 3′-OH but with 4′ additions (4′-C-methyl, 4′-C-ethyl) or sugar ring modifications (d-carba dTTP) were evaluated using pre-steady-state kinetics in low (0.5 mM) and high (6 mM) Mg2+ with HIV reverse transcriptase (RT). Analogues showed diminished observed incorporation rate constants (kobs) compared to dTTP ranging from about 2-fold (3′-H, −N3, and d4TTP with high Mg2+) to >10-fold (3′-NH2 and 3′-F with low Mg2+), while 3′-O-CH3 dTTP incorporated much slower than other analogues. Illustrating the importance of interactions between Mg2+ and the 3′-OH, kobs using 5 μM dTTP and 0.5 mM Mg2+ was only modestly slower (1.6-fold) than with 6 mM Mg2+, while analogues with 3′ alterations incorporated 2.8–5.1-fold slower in 0.5 mM Mg2+. In contrast, 4′-C-methyl and d-carba dTTP, which retain the 3′-OH, were not significantly affected by Mg2+. Consistent with these results, analogues with 3′ modifications were better inhibitors in 6 versus 0.5 mM Mg2+. Equilibrium dissociation constant (KD) and maximum incorporation rate (kpol) determinations for dTTP and analogues lacking a 3′-OH indicated that low Mg2+ caused a several-fold greater reduction in kpol with the analogues but did not significantly affect KD, results consistent with a role for 3′-OH/Mg2+ interactions in catalysis rather than nucleotide binding. Overall, results emphasize the importance of previously unreported interactions between Mg2+ and the 3′-OH of the incoming nucleotide and suggest that inhibitors with 3′-OH groups may have advantages in low free Mg2+ in physiological settings.

Evolution of DNA sequencing.

Sanger and coworkers introduced DNA sequencing in 1970s for the first time. It principally relied on termination of growing nucleotide chain when a dideoxythymidine triphosphate (ddTTP) was inserted in it. Detection of terminated sequences was done radiographically on Polyacrylamide Gel Electrophoresis (PAGE). Improvements that have evolved over time in original Sanger sequencing include replacement of radiography with fluorescence, use of separate fluorescent markers for each nucleotide, use of capillary electrophoresis instead of polyacrylamide gel electrophoresis and then introduction of capillary array electrophoresis. However, this technique suffered from few inherent limitations like decreased sensitivity for low level mutant alleles, complexities in analyzing highly polymorphic regions like Major Histocompatibility Complex (MHC) and high DNA concentrations required. Several Next Generation Sequencing (NGS) technologies have been introduced by Roche, Illumina and other commercial manufacturers that tend to overcome Sanger sequencing limitations and have been reviewed. Introduction of NGS in clinical research and medical diagnostics is expected to change entire diagnostic approach. These include study of cancer variants, detection of minimal residual disease, exome sequencing, detection of Single Nucleotide Polymorphisms (SNPs) and their disease association, epigenetic regulation of gene expression and sequencing of microorganisms genome.

Two X family DNA polymerases, λ and μ, in meiotic tissues of the basidiomycete, Coprinus cinereus

The X family DNA polymerases lambda (CcPollambda) and mu (CcPolmu) were shown to be expressed during meiotic prophase in the basidiomycete, Coprinus cinereus. These two polymerases are the only members of the X family in the C. cinereus genome. The open reading frame of CcPollambda encoded a predicted product of 800 amino acid residues and that of CcPolmicro of 621 amino acid residues. Both CcPollambda and CcPolmicro required Mn(2+) ions for activity, and both were strongly inhibited by dideoxythymidine triphosphate. Unlike their mammalian counterparts, CcPollambda and CcPolmicro had no terminal deoxynucleotidyl transferase activity. Immunostaining analysis revealed that CcPollambda was present at meiotic prophase nuclei in zygotene and pachytene cells, which is the period when homologous chromosomes pair and recombine. CcPolmicro was present in a slightly wider range of cell stages, zygotene to diplotene. In analyses using D-loop recombination intermediate substrates, we found that both CcPollambda and CcPolmicro could promote primer extension of an invading strand in a D-loop structure. Moreover, both polymerases could fully extend the primer in the D-loop substrate, suggesting that D-loop extension is an activity intrinsic to CcPollambda and CcPolmicro. Based on these data, we discuss the possible roles of these polymerases in meiosis.

Nucleotide excision repair functions in the removal of chromium-induced DNA damage in mammalian cells

Some hexavalent chromium (Cr(VI))-containing compounds are human lung carcinogens. While ample information is available on the genetic lesions produced by Cr, surprisingly little is known regarding the cellular mechanisms involved in the removal of Cr-DNA adducts. Nucleotide excision repair (NER) is a highly versatile pathway that is responsive to a variety of DNA helix-distorting lesions. Binary Cr-DNA monoadducts do not produce a significant degree of helical distortion. However, these lesions are unstable due to the propensity of Cr(III) to form DNA adducts (DNA interstrand crosslinks, DNA-protein/amino acid ternary adducts) which may serve as substrates for NER. Therefore, the focus of this study was to determine the role of NER in the processing of Cr-DNA damage using normal (CHO-AA8) and NER-deficient [UV-5 (XP-D); UV-41 (ERCC4/XP-F)] hamster cells. We found that both UV-5 and UV-41 cells exhibited an increased sensitivity towards Cr(VI)-induced clonogenic lethality relative to AA8 cells and were completely deficient in the removal of Cr-DNA adducts. In contrast, repair-complemented UV-5 (expressing hamster XPD) and UV-41 (expressing human ERCC4) cells exhibited similar clonogenic survival and removed Cr-DNA adducts to a similar extent as AA8 cells. In order to extend these findings to the molecular level, we examined the ability of Cr(III)-damaged DNA to induce DNA repair synthesis in cell extracts. Repair synthesis was observed in reactions using extracts derived from AA8, or repair-complemented, but not NER-deficient cells. Cr(III)-induced repair resynthesis was sensitive to inhibition by the DNA polymerase delta/epsilon inhibitor, aphidicolin, but not 2',3'-dideoxythymidine triphosphate (ddTTP), a polymerase beta inhibitor. These results collectively suggest that NER functions in the protection of cells from Cr(VI) lethality and is essential for the removal of Cr(III)-DNA adducts. Consequently, NER may represent an important mechanism for preventing Cr(VI)-induced mutagenesis and neoplastic transformation.

DNA-polymerase α, β, δ and ε activities in isolated neuronal and astroglial cell fractions from developing and aging rat cerebral cortex

The relative proportions of DNA-polymerases α, β, δ and ε (pols α, β, δ and ε) activities in isolated neuronal and astroglial cell fractions from developing, adult and aging rat brain cerebral cortex, were examined. This was achieved through a protocol that takes advantage of the reported differential sensitivities of different DNA-polymerases towards certain inhibitors like butylphenyl and butylanilino nucleotide analogs, 2′,3′-dideoxythymidine triphosphate (ddTTP), monoclonal antibody of human α polymerase and the use of two template primers as substrates. The results indicate that while DNA-polymerase β (pol β) is the predominant enzyme, significant levels of DNA-polymerases α and δ/ε (pols α and δ/ε) are also present in both cell types at all the post-natal ages studied. A notable difference regarding the relative abundance of DNA-polymerases other than β is the higher percentage of pol δ/ε in neurons and a more sustained pol α activity through the life span in astroglia. The presence of detectable proportion of DNA-polymerases other than β (particularly the δ/ε type) may be taken to indicate their role in long patch base excision repair as well as in other modes of DNA repair.

Substitutions of Phe61 Located in the Vicinity of Template 5′-Overhang Influence Polymerase Fidelity and Nucleoside Analog Sensitivity of HIV-1 Reverse Transcriptase

Human immunodeficiency virus type 1 reverse transcriptase (RT) is an error-prone DNA polymerase. Structural determinants of its fidelity are incompletely understood. RT/template primer contacts have been shown to influence its fidelity and sensitivity to nucleoside analog inhibitors. The Phe(61) residue, located within the beta 3 sheet of the finger subdomain, is highly conserved among retroviral RTs. The crystal structure of a ternary complex revealed that Phe(61) contacts the first and second bases of the 5'-template overhang. To determine whether such contacts influence the dNTP-binding pocket, we performed a limited vertical scanning mutagenesis (Phe --> Ala, Leu, Trp, or Tyr) at Phe(61). The F61A mutant displayed the highest increase in fidelity, followed by the F61L and F61W variants, which had intermediate phenotypes. F61Y RT had a minimal effect. The increase in fidelity of the F61A mutant was corroborated by a 12-fold decrease in its forward mutation rate. The Phe(61) mutant RTs also displayed large reductions in sensitivity to 2',3'-dideoxythymidine triphosphate and 2',3'-dideoxy,2'3'-didehydrothymidine triphosphate. Mutants displaying the largest increase in fidelity (F61A and F61L) were also the most resistant. These results suggest that contacts between the finger subdomain of human immunodeficiency virus type 1 RT and the template 5'-overhang are important determinants of the geometry of the dNTP-binding pocket.

Inhibition of adenovirus DNA polymerase by modified nucleoside triphosphate analogs correlate with their antiviral effects on cellular level.

Adenovirus (Ad) infection results in significant morbidity and mortality in both immunocompetent and immunosuppressed hosts. There is currently no licensed chemotherapy effective in dealing with this virus infection. In this study the anti-adenoviral activity of a group of modified nucleoside analogs was investigated. The most efficient 3-fluorosubstituted nucleoside triphosphate inhibitors of Ad DNA polymerase were 3'-fluorothymidine triphosphate (IC50 0.63 microM), 2',3'-dideoxy-3'-fluoroguanosine triphosphate (IC50 0.71 microM) and 2',3'-dideoxy-3'-fluorouridine triphosphate (IC50 2.96 microM). The most efficient 2',3'-dideoxynucleoside triphosphates were 2',3'-dideoxycytidine triphosphate (ddCTP; IC50 1.0 microM), 2',3'-dideoxyadenosine triphosphate (IC50 1.6 microM) and 2',3'-dideoxythymidine triphosphate (IC50 1.82 microM). Kinetic studies indicate competitive inhibition of adenovirus DNA polymerase by ddCTP. These data confirm results previously obtained at the cellular level using a focus reduction assay involving Ad2-infected FL cells. Whereas the D-enantiomers 3'-fluorothymidine and 2',3'-dideoxycytidine are potent inhibitors of adenoviral replication, the corresponding L-enantiomers exhibited no inhibitory activity.

Interaction of tRNA-Derivatives and Oligonucleotide Primers with AZT-Resistant Mutants of HIV-1 Reverse Transcriptase

While the molecular basis of HIV-1 AZT resistance has been widely studied, a biochemical explanation of this process is not well known. No significant changes in the binding affinity of reverse transcriptase (RT) mutants for AZT-triphosphate has been found. Here we analyzed the interaction of wild type and AZT-resistant mutant forms of HIV-1 RT with different primers. Site-directed mutagenesis was used to introduce point mutations on the retroviral enzyme. Primers were either synthetic oligonucleotides or tRNA Lys3 derivatives containing d(pT) n or r(pU) n at the 3′ end. In all cases, determination of kinetic parameters was done in the presence or absence of compounds known to modify protein conformation, such as dimethyl sulfoxide (DMSO), urea, and Triton X-100. Although we found similar K m values for all RTs, there was generally an increase in the affinity when enzymes were tested in the presence of DMSO, urea, and Triton X-100. Then, we analyzed the nucleation and elongation steps of the polymerization process. The efficiency of formation of the first base pair was determined by measuring K m1, the affinity between RT and the 3′ terminal nucleotide of the primer. An important difference was found: in the presence of DMSO, urea, and Triton X-100, the K m1 values for mutated enzymes were higher than those of wild type RTs. Thus, the presence of compounds able to change protein conformation led to a marked destabilization of the interaction of mutated RTs with the 3′ terminal nucleotide of the primer. From these results, it can be hypothesized that resistance to AZT is not due to the direct influence of mutations on RT, but rather to conformational changes of the mutated RT in complex with the template–primer altering the ability of the enzyme to select or reject an incoming dNTP. 1 Abbreviations: AIDS, acquired immune deficiency syndrome; HIV-1, human immunodeficiency virus type-1, RT, reverse transcriptase; AZT, 3′-azido-2′,3′-dideoxythymidine; AZT-TP, 3′-azido-2′,3′-dideoxythymidine triphosphate; dNTP, deoxynucleoside triphosphates; ddNTP, dideoxynucleoside triphosphates; DMSO, dimethyl sulfoxide. 1

Purification and characterization of a 100 kDa DNA polymerase from cauliflower inflorescence.

A DNA polymerase from cauliflower (Brassica oleracea var. botrytis) inflorescence has been purified to near homogeneity through five successive column chromatographies, and temporally designated cauliflower polymerase 1. Cauliflower polymerase 1 is a monopolypeptide with a molecular mass of 100 kDa. The enzyme efficiently uses synthetic DNA homopolymers and moderately activated DNA and a synthetic RNA homopolymer as template-primers. The enzyme is strongly sensitive to dideoxythymidine triphosphate and N-ethylmaleimide, but it is insensitive to aphidicolin. It was stimulated with 250 mM KCl. Its mode of DNA synthesis is high-processive with or without proliferating-cell nuclear antigen. A 3'-->5' exonuclease activity is associated with cauliflower polymerase 1. The enzyme is clearly different from cauliflower mitochondrial polymerase and does not resemble the four different types of wheat DNA polymerase, designated wheat DNA polymerases A, B, CI and CII. In the present paper the role of the enzyme in plant DNA synthesis is discussed.

Characterization of the Reverse Transcriptase of a Human Immunodeficiency Virus Type 1 Group O Isolate

The catalytic properties and sensitivity to different inhibitors have been determined for the reverse transcriptase (RT) of group O human immunodeficiency virus type 1 (HIV-1). The RT-coding region was cloned from a new HIV-1 group O isolate from Spain, expressed inEscherichia coli,and purified by affinity chromatography. This new RT showed 79% amino acid sequence identity with the corresponding enzyme of group M subtype B strain BH10. The two enzymes showed very similar kinetics of RNA-dependent DNA polymerization using homopolymeric template-primers and RNase H specific activity. Inhibitor sensitivity to ddTTP and 3′-azido-2′,3′-dideoxythymidine triphosphate (AZTTP) was also similar for both enzymes. However, the two enzymes differed dramatically in their sensitivity to several inhibitors. While the RT of the BH10 isolate was sensitive to nevirapine and loviride (IC50ranged from 0.16 to 8.2 μM,depending on the substrates used), the enzyme of the Spanish HIV-1 group O isolate showed high-level resistance to those compounds (IC50> 200 μM). The amino acid sequence of the RT of group O HIV-1 contains three amino acids (Cys-181, Glu-179, and Gly-98), which are found in group M subtype B strains resistant to nonnucleoside RT inhibitors. The recombinant group O HIV-1 RT should be useful for studies aimed at discovering and designing drugs directed toward group O isolates of HIV-1.

DNA polymerases δ and e in developing and aging rat brain

Our study reveals the presence of DNA polymerases δ and e, participating in DNA replication and repair, along with already known polymerases α and β, in the developing and aging rat brain. This was achieved through a protocol that takes advantage of the reported differential sensitivities of different DNA polymerases towards certain inhibitors such as butylphenyl and butylanilino nucleotide analogs, 2′, 3′-dideoxythymidine triphosphate, the monoclonal antibody of human polymerase α and the use of preferred template primers and proliferating cell nuclear antigen. The results indicate that while polymerase β seems to be the predominant one, significant levels of polymerases α, δ and e are also present at all the postnatal ages studied and that the relative proportion of polymerase e increases with age. The data suggest that the rat brain is equipped with a sustained DNA repair capacity throughout the life span.

KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells.

Administration of exogenous keratinocyte growth factor (KGF) prevents or attenuates several forms of oxidant-mediated lung injury. Because DNA damage in epithelial cells is a component of radiation pneumotoxicity, we determined whether KGF ameliorated DNA strand breaks in irradiated A549 cells. Cells were exposed to 137Cs gamma rays, and DNA damage was measured by alkaline unwinding and ethidium bromide fluorescence after a 30-min recovery period. Radiation induced a dose-dependent increase in DNA strand breaks. The percentage of double-stranded DNA after exposure to 30 Gy increased from 44.6 +/- 3.5% in untreated control cells to 61.6 +/- 5.0% in cells cultured with 100 ng/ml KGF for 24 h (P < 0.05). No reduction in DNA damage occurred when the cells were cultured with KGF but maintained at 0 degree C during and after irradiation. The sparing effect of KGF on radiation-induced DNA damage was blocked by aphidicolin, an inhibitor of DNA polymerases-alpha, -delta, and -epsilon and by butylphenyl dGTP, which blocks DNA polymerase-alpha strongly and polymerases-delta and -epsilon less effectively. However, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerase-beta, did not abrogate the KGF effect. Thus KGF increases DNA repair capacity in irradiated pulmonary epithelial cells, an effect mediated at least in part by DNA polymerases-alpha, -delta, and -epsilon. Enhancement of DNA repair capability after cell damage may be one mechanism by which KGF is able to ameliorate oxidant-mediated alveolar epithelial injury.

Identification of the antiviral nucleoside drug binding site of HIV-1 integrase by proteolytic peptide mapping: R. Drake, N. Neamati, P. Sunthankar, A. Mazumder, Y. Pommier. Dept. of Biochemistry, Univ. of Arkansas for Med. Sci., Little Rock, AR 72205 and Laboratory of Mol. Pharm., NCI, NIH, Bethesda, MD, 20892

Carbocylic 2′,3′-didehydro-2′,3′-dideoxyguanosine (Carbovir; NSC 614846) is an antiretroviral agent which may be useful in the treatment of AIDS. We have synthesized the 5′-triphosphate of Carbovir and examined its ability to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (EC 2.7.7.49) and other retroviral reverse transcriptases, as well as human DNA polymerases α, β, γ (EC 2.7.7.7) and DNA primase (EC 2.7.7.6). Carbovir triphosphate emerges as a highly selective inhibitor of reverse transcriptases with little, if any, effect on the cellular enzymes. 3′-Azido-2′,3′-dideoxythymidine (AZT) triphosphate and the two dideoxynucleoside triphosphates, ddTTP and ddGTP, inhibited HIV-1 reverse transcriptase to the same degree as Carbovir triphosphate, but were less selective in that they also inhibited DNA polymerases β and γ. We conclude that Carbovir is a highly selective antiretroviral agent.

A DNA Polymerase α Catalytic Subunit Is Purified Independently from the Tissues at Meiotic Prometaphase I of a Basidiomycete,Coprinus cinereus

A 135kDa DNA polymerase α lacking primase activity has been purified to near homogeneity fromCoprinusmeiotic tissues. The activity of the DNA polymerase was sensitive to aphidicolin and N-ethylmaleimide, but was insensitive to dideoxythymidine triphosphate. DNA synthesis was proceeded with a low processivity. Neither activity nor processivity were affected by PCNA in the presence or absence of KCl. Monovalent cation inhibited its activity. These biochemical properties are almost identical to those ofCoprinusDNA polymerase α -primase complex. However, the135kDa DNA polymerase did not use activated DNA as a template-primer, inconsistent withCoprinusDNA polymerase α-primase complex. The 135kDa DNA polymerase was purified from the tissues at meiotic pro-metaphase I, suggesting that the α- DNA polymerase -primase complex dissociates as the meiotic cell cycle progresses and only the catalytic subunit remains at this stage.

Purification and characterization of a β-like DNA polymerase from Trypanosoma cruzi

A DNA polymerase was purified to near homogeneity from Trypanosoma cruzi epimastigotes. This preparation had a major polypeptide of 50 kDa and a minor band of 45 kDa. SDS-PAGE studies and a novel colorimetric activity gel technique demonstrated that the 50-kDa polypeptide chain is the catalytic subunit of this T. cruzi DNA polymerase. Western blot analysis of different purification stage fractions strongly suggests that this 50-kDa protein is the intact catalytic subunit and does not correspond to a degradation product from a larger one. This T. cruzi DNA polymerase is insensitive to aphidicolin, butylphenyldeoxyguanosine triphosphate, berenil, ethidium bromide and N-ethylmaleimide, but is markedly inhibited by the dideoxythymidine triphosphate analogue. Studies with different DNA templates showed that the DNA polymerase prefers activated DNA as substrate and that it cannot elongate oligoriboadenylate primers. The data presented in this paper are consistent with the hypothesis that this enzyme corresponds to a β-like DNA polymerase present in the parasitic protozoon T. cruzi.

Human Immunodeficiency Virus Type 1 Reverse Transcriptase: 3′-AZIDODEOXYTHYMIDINE 5′-TRIPHOSPHATE INHIBITION INDICATES TWO-STEP BINDING FOR TEMPLATE-PRIMER

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) catalyzes DNA synthesis by an ordered sequential mechanism. After template-primer (T.P) binds to free enzyme, the deoxynucleoside triphosphate to be incorporated binds to the RT and T.P binary complex (RTT.P). After incorporation of the bound nucleotide, catalytic cycling is limited either by a conformational change (for processive synthesis) or release of the enzyme from the extended T.P (for single-nucleotide incorporation). To explore cycling through these alternate rate-limiting steps, we determined kinetic parameters for single-nucleotide incorporation by HXB2R HIV-1 RT with chain-terminating nucleotide substrates 3'-azido-3'-deoxythymidine triphosphate (AZTTP) and dideoxythymidine triphosphate on a homopolymeric T.P system, poly(rA)-oligo(dT)16. Inhibition of processive deoxythymidine monophosphate incorporation by these chain-terminating substrates was also examined. Because AZTTP is a substrate, its Km should be equivalent to Ki, and since Km for AZTTP should be influenced by the dissociation rate constant for RTT.P, we examined the effect of altering RTT.P dissociation on AZTTP kinetic parameters. The dissociation rate constant was modulated by making use of different T.P substrates, viral sources of RT, and a mutant RT altered at a residue that perturbs T.P binding. As expected from earlier work, the time course of AZTMP incorporation on poly(rA)-oligo(dT)16 was biphasic, with a burst followed by a slower steady-state phase representing kcat (0.42 min-1) which was similar to the rate constant for RTT.P dissociation. Additionally, Km for AZTTP (110 nM) was lower than its equilibrium dissociation constant (1200 nM). AZTTP inhibition (Ki,AZTTP) of processive dTMP incorporation and incorporation of a single nucleotide were similar. However, a simple correlation between the RTT.P dissociation rate constant and Ki,AZTTP was not observed. These results indicate that a simple ordered model for single-nucleotide incorporation is inadequate and that different forms of RTT.P exist which can limit catalysis. The results are discussed in the context of a two-step binding reaction for T.P where the binary RTT.P complex undergoes an isomerization before binding of the deoxynucleotide substrate.

Effect of DNA polymerase inhibitors on repair of γ ray-induced DNA damage in proliferating (intact versus permeable) human fibroblasts: evidence for differences in the modes of action of aphidicolin and 1-β- d-arabinofuranosylcytosine

The mammalian DNA polymerase inhibitors aphidicolin and 1-β- d-arabinofuranosylcytosine (araC), when used in combination, inhibit the repair of DNA damage induced by γ rays or 4-nitroquinoline 1-oxide in normal human fibroblasts to an extent 2- to 4-fold greater than that seen with each inhibitor alone. Thus either modulates the rate of intracellular accumulation of araC 5′-triphosphate (araCTP), the presumed rate-limiting step in genotoxic action of araC, or aphidicolin and araC inhibit repair by different mechanisms. To explore these possibilities, we compared the effects of aphidicolin, araC, araCTP, and 2′,3′-dideoxythymidine triphosphate (ddTTP) on repair of DNA damage induced by 60Co γ radiation in intact versus permeable human fibroblasts. Both aphidicolin and araC strongly inhibited repair in permeable cells, as indicated by the accumulation of DNA strand breaks in irradiated cultures that were subsequently treated with saponin (25 μg/ml; 10 min) and incubated for 2 h with either chemical. The extent of repair inhibition by each drug was comparable in intact and permeable cells, amounting to ∼ 1.1 sites/10 8 daltons/2 h upon exposure to upon Gy. The active metabolite of araC, araCTP, did not inhibit repair in intact cells, but did so in permeable cells to an extent within the range of that seen with araC or aphidicolin alone. The incidence of DNA strand breaks accumulating in γ-irradiated permeable cultures as a result of incubation with araCTP plus aphidicolin, or araC plus aphidicolin, was ∼ 2-fold greater than that arising in parallel cultures which had been incubated with optimal concentrations of each of the three drugs alone. Although the resolution of our assays compelled us to monitor repair events in moribund cell populations, we have reason to be confident that within the short post-irradiation period considered here, the observed drug-accumulated breaks truly represent functional repair inhibition adn not merely abortive pathological responses. We thus conclude that (1) the accumulation of araCTP in intact cells is not limiting the ability of araC to inhibit DNA repair; and (2) the mode of the inhibitory action of araC/araCTP on γ ray repair is different from that of aphidicolin. In constrast to the observations with these chemicals, ddTTP (20 μM), a potent inhibitor of DNA polymerase β, did not produce any measurable effect on DNA repair in γ-irradiated permeable fibroblasts, nor did it enhance the efficacy of araC, araCTP or aphidicolin to inhibit repair. These results strongly suggest that DNA polymerase β plays no significant role in the repair of γ radioproducts in human fibroblasts. The permeabilized cell system adopted here does not adversely influence the repair capability of human fibroblasts and may thus prove useful in studying the enzymology of DNA repair in proliferating cultures following exposure to ionizing radiation and other genotoxic agents.

Subunit-selective mutagenesis of Glu-89 residue in human immunodeficiency virus reverse transcriptase. Contribution of p66 and p51 subunits to nucleoside analog sensitivity, divalent cation preference, and steady state kinetic properties.

The E89G alteration in the human immunodeficiency virus type 1 reverse transcriptase has been shown to confer resistance to nucleoside analogs and a loss of magnesium cation preference (Prasad, V.R., Lowy, I., De Los Santos, T., Chiang, L., and Goff, S.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11363-11367. The wild type reverse transcriptase heterodimer, chimeric reverse transcriptases that contain the E89G alteration in one of the subunits (p66wt/p51m and p66m/p51wt), and the mutant enzyme (p66m/p51m) were prepared. Analysis of steady state kinetic parameters showed that the mutant enzyme (p66m/p51m) displayed a higher Vmax, a higher Km for 2'-deoxythymidine triphosphate, and a higher Ki for 2',3'-dideoxythymidine triphosphate than the wild type enzyme. The increased Km and Ki values were observed only when a heterodimer contained the alteration in the p66 subunit. Tests for divalent cation requirement showed that only the dimers containing the wild type p66 (p66wt/p51wt and p66wt/p51m) displayed a preference for magnesium. Our results indicate that p66 plays a dominant role in deoxynucleotide triphosphate substrate recognition (Km), nucleoside analog sensitivity (Ki), and magnesium preference. However, the increased Vmax displayed by the mutant enzyme (p66m/p51m) appeared to be determined by both of the subunits.

Reverse transcriptase activity of an intron encoded polypeptide.

A number of group II introns from eukaryotic organelles and prokaryotes contain open reading frames for polypeptides with homology to retroviral reverse transcriptases (RTs). We have used the yeast transposon (Ty) system to express ORFs for RTs from eukaryotic organelles. This includes the mitochondrial coxI intron i1 from the fungus Podospora anserina, the plastid petD intron from the alga Scenedesmus obliquus and the mitochondrial RTL gene from the alga Chlamydomonas reinhardtii. The ORFs were fused with the TYA ORF from the yeast retrotransposon Ty to produce virus-like particles in the recipient strains with detectable amounts of the RT-like polypeptides. Analysis of the heterologous gene products revealed biochemical evidence that the P. anserina intron encodes an RNA-directed DNA polymerase with properties typically found for RTs of viral or retrotransposable origin. In vitro assays showed that the intron encoded RT is sensitive to RT inhibitors such as N-ethylmaleimide and dideoxythymidine triphosphate but is insensitive against the DNA polymerase inhibitor aphidicolin. The direct biochemical evidence provided here supports the idea that intron encoded RTs are involved in intron transposition events.

Strand displacement activity of the human immunodeficiency virus type 1 reverse transcriptase heterodimer and its individual subunits.

By using a DNA substrate with defined gap size, we found that human immunodeficiency virus type 1 reverse transcriptase (HIV-RT) was able to perform strand displacement DNA synthesis. This activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor C and second by Escherichia coli single-stranded DNA-binding protein, which together allow DNA polymerase delta to perform strand displacement DNA synthesis (Podust, V., and Hübscher, U. (1993) Nucleic Acids Res. 21, 841-846). 3'-Azido-2',3'-dideoxythymidine triphosphate inhibited displacement completely, indicating that DNA synthesis is required for this reaction. The HIV-RT p66 polypeptide alone could perform limited strand displacement DNA synthesis, whereas the HIV-RT p51 polypeptide was completely inactive, likely due to its inability to replicate extensively on a M13 DNA template. On the other hand the HIV-RT p51 polypeptide enhanced the strand displacement activity of the HIV-RT p66 subunit at a molar ratio of 4:1, mainly by chasing short products into longer ones. Furthermore, kinetic experiments after complementation of HIV-RT p66 with HIV-RT p51 indicated that HIV-RT p51 can restore rate and extent of strand displacement activity by HIV-RT p66 compared with the HIV-RT heterodimer p66/p51, suggesting a function of the 51-kDa polypeptide.