Levels Of DNA Polymerase Research Articles

Age-related changes in DNA polymerase α expression

DNA polymerase α isozymes differing in specific activity and affinity of binding to DNA were purified from human fibroblasts derived from donors of different ages. Fetal-derived fibroblasts expressed a single, high-activity enzyme (A 2), with high affinity of binding to DNA. Adult-derived fibroblasts exhibited two forms of DNA polymerase α, one identical to the fetal enzyme, and a second with about tenfold less activity showing low affinity of binding to DNA (A 1). The ratio of DNA polymerase A 2/A 1 decreased dramatically with age from 100° A 2 in fetal-derived fibroblasts to about 94° A 1 in fibroblasts derived from a 66-year-old donor. The DNA binding affinity of polymerase α A 1 from adult-derived fibroblasts increased concominant with a significant increase in activity when the enzyme was treated with phosphatidylinositol-4-monophosphate (PIP), or with inositol-1, 4-bisphosphate (I(1,4)P 2). The enzyme reverted back to a less active form, with loss of the noncovalently bound I(1,4)P 2, as a function of time. When permeabilized human fibroblasts with low DNA excision repair capacity were treated with 7,8-dihydrodiol-9,10-epoxybenzo(a)-pyrene (BPDE) in the presence of 32P-ATP, phosphatidylinositol, and cycloheximide, excision repair was initiated and 32P-labeled DNA polymerase α was recovered in the absence of de novo protein synthesis. DNA synthesis associated with either scheduled DNA synthesis or BPDE-initiated excision repair declined as a function of increased age in human cells. The data suggest that the decline in both DNA excision repair-associated and mitogen-activated DNA synthesis may be correlated with decreased total intracellular levels of DNA polymerase and with the decline in polymerase α activity as a function of age, that DNA repair-associated initiation of DNA synthesis in adult-derived cells may increase with activation of a pool of low activity DNA polymerase α, and that DNA polymerase α activity increases as a function of enzyme interaction with a component of the PI phosphorylation cascade.

DNA polymerase β in human thyroid of Graves' disease and thyroid tumors

We have shown that the level of DNA polymerase β of rat adrenal cortex is regulated by pituitary trophic hormones and may correlate with their endocrine function. Here we measured DNA polymerase β activity in human thyroid tissues of various benign and malignant thyroid disorders in order to verify the correlation between DNA polymerase β activity and endocrine function. In Graves' disease (hyperfunction), the level of DNA polymerase β per cell was three times higher than in normal thyroid, while in undifferentiated thyroid carcinomas this enzyme level was lower than normal. Furthermore, DNA polymerase β in the crude extracts of cancer cells showed larger molecular forms, ranging from five to 12S, upon sucrose gradient sedimentation. These observations further support the hypothesis that the activity of DNA polymerase β correlates, in part, with the functional level of the endocrine organ and with cell differentiation.

Specific inhibition of DNA biosynthesis induced by 3'-amino-2',3'-dideoxycytidine.

3'-Amino-2',3'-dideoxycytidine (3'-NH2-dCyd) produced an S-phase-specific block in exponentially growing L1210 leukemia cells. The monophosphate and triphosphate forms of the drug were detected within a few hours of 3'-NH2-dCyd treatment of intact cells. No significant change in the deoxynucleoside triphosphate levels was observed during the early stages of treatment. However, by 24 h a 2-fold increase in the amount of the deoxynucleoside triphosphates was seen. The triphosphate form of the drug competitively inhibited dCTP incorporation into calf thymus DNA using highly purified DNA polymerase alpha. The Ki was determined to be 9.6 microM with respect to dCTP. Incorporation of the analogue into DNA was not detected. On the other hand, sucrose gradient analysis suggested that incorporation of the analogue into actively synthesized DNA may account for the biological activity of this compound. Treatment with 3'-NH2-dCyd induced single-strand breaks in actively synthesized DNA, but no double-strand breaks were observed in the presence of the analogue. The data indicate that 3'-amino-2',3'-dideoxycytidine specifically interferes with DNA replication at the level of DNA polymerase by inhibiting chain elongation.

Hormonal regulation of DNA polymerase-beta activity in the rat thyroid gland.

Using hypophysectomized rats, it has been shown that DNA polymerase-beta activity in the adrenal gland and testis is largely influenced by pituitary trophic hormones. Sucrose gradient centrifugation of thyroid extracts revealed three peaks of DNA polymerase-beta activity sedimenting at 3.3S, 7.3S and 12S. Of these, hypophysectomy induced a decrease in the 3.3S DNA polymerase-beta, whereas other molecular forms were affected only slightly. DNA polymerase-alpha and -gamma activities were unaffected by hypophysectomy. These changes in DNA polymerase-beta caused by hypophysectomy were reversed by daily i.p. injection of TSH. Furthermore, stimulation of the thyroid by excess TSH induced by the administration of 1-methyl-2-mercaptoimidazole resulted in an increase of all forms of thyroid DNA polymerase-beta. These results show that the level of DNA polymerase is relatively constant after hypophysectomy but that DNA polymerase-beta in the rat thyroid gland is also modulated by TSH mainly through the change of activity of the polymerase-beta which sediments at 3.3S.

Antibodies to Epstein-Barr Virus—Specific DNase and DNA Polymerase in the Chronic Fatigue Syndrome

In an attempt to examine further the association between active Epstein-Barr virus (EBV) infection and the chronic fatigue syndrome (chronic EBV syndrome, or chronic or atypical mononucleosis), antibodies acting against EBV-specific DNase and DNA polymerase, which are expressed only during virus replication, were assayed. Serum samples from 25 healthy EBV-seropositive individuals neutralized 3.5 +/- 5.1 U (mean +/- SD) of DNase activity and 14.7 +/- 8.5 U of DNA polymerase activity. From these values were selected upper limits of anti-EBV enzyme activity of 17.9 and 31.3 U neutralized in normal individuals, respectively (representing the 95% confidence limit). Serum samples from six groups of subjects representing a variety of EBV-related illnesses were then studied. Only patients with notably elevated anti-EBV antibody titers to viral capsid antigen (VCA) (greater than 10,000) had elevated levels of anti-EBV DNase (38 to 56 U neutralized) and anti-EBV DNA polymerase (72 to 106 U neutralized). Three additional patients and two geriatric controls with average anti-EBV early antigen/VCA titers had slightly elevated levels of antibody to EBV DNA polymerase. IgA anti-VCA, anti-early antigen antibodies, or both, were also detected in the same patients who had high EBV DNase and polymerase antibody levels. These antibody profiles are similar to those in patients with nasopharyngeal carcinoma. Since three of the six patients with elevated anti-EBV enzyme antibody levels developed fatal lymphomas, patients with chronic EBV and this antibody profile might be in another illness category at risk for malignant disease.

DNA polymerase I activity in Escherichia coli is influenced by spot 42 RNA

We have shown that the level of DNA polymerase I (Pol I) activity in Escherichia coli is influenced by the level of a 109-nucleotide RNA, spot 42 RNA. Deletion of the gene for spot 42 RNA results in a 20 to 25% decrease in Pol I activity, as assayed by nucleotide incorporation in cell extracts and a decrease in the ability of cells to grow in the presence of the DNA-alkylating agent methyl methanesulfonate. Also, a physiological reduction of the level of spot 42 RNA, by growth in media containing poor carbon sources, results in a corresponding decrease in Pol I activity. Conversely, overproduction of spot 42 RNA results in a 10 to 15% increase in Pol I activity in vitro. Thus, changes in the amount of spot 42 RNA result in relatively small but significant changes in Pol I activity.

Experimental duck hepatitis B virus infection: pathology and evolution of hepatic and extrahepatic infection.

Seventy, 1-day-old ducklings inoculated intraperitoneally with duck hepatitis B virus and 30 controls have been studied over a 2-year period. Infection with duck hepatitis B virus occurred in all inoculated ducks, although this was not associated with clinical morbidity. Duck hepatitis B virus DNA was first detected in liver on Day 3, in pancreatic acinar cells on Day 4, serum on Day 6, splenic red and white pulp on Day 7 and in the renal glomurulus on Day 14, using a combination of dot, Southern blot and in situ hybridization techniques. Peak levels of circulating virus, as determined by DNA polymerase levels, occurred 1 to 4 weeks postinoculation. Mild degrees of portal inflammation were seen in sections of liver tissue in both infected and control ducks. However, moderately severe inflammatory changes were present in 8 of 22 infected birds compared with 0 of 18 controls (p less than 0.025). Appearance of this inflammatory infiltrate 6 weeks postinoculation coincided with a decrease in levels of duck hepatitis B virus DNA in hepatocytes and within the pancreatic acinar cells. At the same time, duck hepatitis B virus DNA became increasingly localized to the splenic germinal centers, and viral DNA was first detected in pancreatic islet cells. No histological changes accompanied the extra-hepatic tissue infection. The sequence and significance of duck hepatitis B virus infection in liver and extra-hepatic tissues is discussed in relation to the pathogenesis of hepatitis B virus infection in man.

Pilot study of recombinant human interleukin 2 for chronic type B hepatitis.

Recombinant human interleukin 2 was administered to 10 patients with chronic type B hepatitis as a part of a pilot study to evaluate its antiviral activity. Patients received 1 to 3 x 10(5) units per day of interleukin 2 for 21 to 28 days, and all completed the treatment schedule. During therapy, serum values of DNA polymerase decreased in 6 and became negative in four patients. However, when therapy was discontinued, DNA polymerase levels increased to pretreatment levels in most cases. Serum HBeAg levels did not change during treatment. Serum aminotransferase levels transiently increased in 6 of the 10 patients during therapy; but once therapy was stopped, levels fell markedly. Side effects of interleukin 2 therapy included fever, chills, anorexia and fatigue. After 1 year of follow-up, three treated patients had lost HBeAg and had marked improvement in aminotransferase levels. These serologic and biochemical improvements occurred 1.5 to 11 months after therapy was stopped. Whether a 3- to 4-week course of interleukin 2 therapy leads to an increased rate of seroconversion from HBeAg to antibody in chronic type B hepatitis deserves further evaluation in prospectively randomized, controlled trials.

Characterization of DNA polymerase α activity from a mouse DNA temperature-sensitive mutant, strain tsFT20, which shows a defect in DNA polymerase α activity at restrictive temperatures

tsFT20 cells derived from mouse FM3A cells are DNA temperature-sensitive mutants, which have heat-labile DNA polymerase α activity. When tsFT20 cells were incubated at restrictive temperatures, intracellular levels of DNA polymerase α activity changed biphasically, showing an initial fast decrease (phase I) and a subsequent slow decrease (phase II). The activity of DNA polymerase α from tsFT20 cells cultured at a permissive temperature (33 °C) was greatly increased by the addition of glycerol or ethylene glycol to the reaction mixture, while little increase in enzyme activity was observed at any concentration of glycerol or ethylene glycol tested with the enzyme from the cells cultured at a restrictive temperature (39 °C) for 8 h (phase II). The activity of DNA polymerase α from wild-type cells was also increased by the addition of glycerol but the increase was much less than that in the tsFT20 cells. An in vitro preincubation experiment showed that DNA polymerase α from tsFT20 cells cultured at 33 °C very rapidly lost its ability to be stimulated by glycerol. Furthermore, the experiment using the extracts prepared from tsFT20 cells cultured at 39 °C for various periods showed that the ability to be stimulated by glycerol decreased with the duration of incubation time at 39 °C. DNA polymerase α from the revertants, which can grow at 39 °C and exhibit a partial recovery in heat stability of DNA polymerase α activity, showed an intermediate response to glycerol, between those of DNA polymerase α from tsFT20 and from the wild-type cells. Finally, it was observed that the level of enzyme activity that can be stimulated by glycerol correlated well with the DNA synthesizing ability of tsFT20 Cells.

Establishment and biological characterization of an in vitro human cytomegalovirus latency model

In an attempt to develop an in vitro human cytomegalovirus (HCMV) latency model system, the growth characteristics of HCMV in a human thyroid papillary carcinoma cell line (TPC-1) were examined. When TPC-1 cultures preheated at 40.5° for 48 hr were infected with HCMV and incubated at a supraoptimal temperature (40.5°), the cultures could be maintained for at least 65 days without detection of infectious virus. In contrast, when the infected cultures were incubated at 37°, HCMV persistently infected cultures were established. HCMV was reactivated from the latently infected cultures by decreasing the incubation temperature from 40.5 to 37°, and the cultures subsequently entered into virus persistent infection. Although HCMV-specific polypeptides which comigrate with the immediate early virus polypeptides and nuclear antigens were continuously detectable in the majority (more than 95%) of the cells during the latent period, a detectable level of virus-specified DNA polymerase (one of the early virus proteins) was not induced, suggesting that the blockage of HCMV replication in the latently infected cultures occurs at the early stages of the HCMV replication cycle. Infectious center assay revealed that 0.002 to 0.2% of the cells contain an HCMV genome that can be activated during the latent period. The latently infected cells were susceptible to superinfection with homologous and heterologous strains of HCMV. In persistently infected cultures approximately 38% of the cells were lysed by reaction with HCMV immune serum and complement, whereas complement-mediated immune cytolysis could not be detected in the latently infected cultures. The data presented suggest that a temperature-sensitive cellular function(s) that controls the expression of the HCMV early functions plays an important role in maintenance of the HCMV genome in the latent state and reactivation of HCMV by decreasing the incubation temperature.

Peroxidase-anti-peroxidase detection of hepatitis B surface and core antigen in liver biopsy specimens from patients with chronic type B hepatitis.

Liver biopsy specimens from 58 American patients with chronic type B hepatitis were investigated for the presence and distribution of the hepatitis B core (HBcAg) and surface (HBsAg) antigens by peroxidase-anti-peroxidase techniques. HBsAg was detected in 43 (77%) and HBcAg in 52 (90%) patients. HBcAg was present in 50 of 51 (98%) patients with hepatitis B e antigen (HBeAg) but in only two of seven (29%) of patients with antibody to HBeAg (anti-HBe). There was no correlation between severity of hepatitis or height of aminotransferase activities and the amount of HBsAg or HBcAg in hepatocytes but there was a positive correlation between amount of HBcAg and height of HBV-DNA and DNA polymerase activity in serum. Follow-up liver biopsies, taken 1 to 3 yr later, were available from 39 patients. HBcAg remained detectable in 25 of 26 patients with persistence of HBeAg but disappeared in 12 patients who had lost HBeAg. In nine patients, HBcAg was cytoplasmic as well as nuclear in distribution. Seven of these patients had an intense lobular hepatitis with marked elevations in aminotransferase activities. These findings indicate that the amount of HBcAg in liver correlates with the amount of serum hepatitis B virus as quantified by serum levels of DNA polymerase and HBV-DNA. The amount of nuclear HBcAg does not correlate with the severity of the liver disease, but the presence of cytoplasmic HBcAg usually reflects an active and severe ongoing hepatitis.

Spot 42 RNA of Escherichia coli is not an mRNA

Spot 42 RNA of Escherichia coli, a 109-nucleotide RNA that influences the level of DNA polymerase I, has an AUG triplet preceded by a purine-rich potential ribosome-binding site and is followed by a short (14-triplet) potential open reading frame. Although the RNA bound to ribosomes, it did so inefficiently and nonproductively. When fused to lacZ sequences, spot RNA did not support the synthesis of beta-galactosidase. Also, the biological effects of spot 42 RNA were not altered by mutation of the tyrosine UAU codon to the chain termination UAG. We conclude that the effects of spot 42 RNA are mediated by the RNA itself and not by a spot 42 RNA-encoded peptide.

A pilot study of BW A515U (6-deoxyacyclovir) in chronic hepatitis B virus infection

BW A515U (6-Deoxyacyclovir) is a pro-drug of acyclovir and almost 100% is absorbed orally. 250 mg orally 6-hourly for 10 days was given to 4 hepatitis B surface antigen/e antigen-positive carriers. No consistent effect on productive viral replication, as determined by serum DNA polymerase and DNA levels was observed. The changes that occurred in these markers and transaminases in 1 patient were attributed to a spontaneous depression of productive viral replication.

Overlapping arrangement of the recF and dnaN operons of 5Escherlchia coli; positive and negative control sequences

The recF gene of Escherichia coli controls one of the recombination pathways and UV sensitivity, but its precise function and expression pattern are still largely unknown. We have characterized the promoter region of the recF gene by mapping for E. coli RNA polymerase binding sites, in vitro transcription experiments, cloning, and S1 mapping of in vivo mRNAs. It contains three overlapping promoters, two initiating transcription towards recF and one in the opposite direction. The recF promoter region is located about 600 bp upstream from the start codon of the recF structural gene and resides entirely within the translated region of the preceding gene, dnaN, which encodes for the β subunit of DNA polymerase III. This unusual arrangement might provide discoordinate regulation of the recF and dnaN genes, thus controlling the level of DNA polymerase III holoenzyme. Expression of recF is also negatively controlled by sequences located upstream as well as inside the recF coding frame. Such negative regulation may serve to prevent toxic effects due to accumulation of an excessive number of copies of the recF gene product.

An open study of human lymphoblastoid interferon and oral acyclovir in chronic hepatitis B virus infection

Ten patients were entered into an open study of interferon (IFN) 'induction' and oral acyclovir (ACV) 'maintenance' therapy. They received 5 Mega units (Mu)/m2 IFN by intramuscular injection daily for 3 days, followed by 7.5 Mu/m2 IFN daily for 7 days. IFN therapy was then discontinued and a 6-week course of oral ACV at a dose of 800 mg 4 times daily commenced. At 6 months, 2 patients had become HBeAg-negative and 1 had developed anti-HBe. Elimination of HBeAg in these patients was accompanied by return of serum liver function tests to normal. There was a statistically significant inhibition of DNA polymerase levels after the 1st week of IFN therapy, which then slowly increased to pretreatment values over 8 weeks. There were no significant adverse effects of ACV therapy, while fever, 'flu-like illness', fatigue, anorexia, and leucopenia were the main side-effects observed during the course of IFN which necessitated dose reduction in 7 patients. Combination therapy appears to effectively inhibit viral replication, although the 'maintenance' effect of oral ACV is minimal. A more effective drug to combine with IFN is needed.

Frameshift mutagenesis by ultraviolet and γ radiations in Salmonella: SOS inducibility and effects of DNA polymerase I

Ultraviolet (UV) and γ-induced mutagenesis have been studied using a doubly auxtrophic strain of Salmonella typhimurium carrying the ambeer leuA150 mutation (which reverts by base-pair substitution) and the frameshift hisC3076 marker (which reverts by compensating frameshifts). In the initially constructed LT2 background, both markers were poorly revertible by UV and essentially by γ-radiation. A derivative of this strain carrying the mutation-enhancing plasmid pKM101 was however readily reverted by both UV and γ, with either Leu + (base substitution) or His + (frameshift) revertants being observed on appropriate selective media. Photoreactivation experiments suggested that the lesions leading to formation of the two types of mutagenic event were similar if not identical. Support for this suggestion was obtained when it was found that yields of both types of UV-induced revertant were significantly increased in an excision-deficient background, while no revertants of either type were found in a recA background. Yields of γ-induced revertants were not greatly altered in a uvrB background, but were also reduced to zero (for both markers) in the recA background. These results are consistent with what has previously been well-documented for UV and γ-induced base-pair substitution mutagenesis, and serve to emphasize the similarities between base-pair substitution mutagenesis and frameshift mutagenesis by these agents. There are differences, however, since although UV-induced reversion of the leuA150 marker was little affected and γ-induced reversion of leuA150 was somewhat reduced in the presence of a polA mutation ( polA3), the yields of His + frameshift revertants were significantly increased in the polA3 background following treatment with either UV or γ. Thus while inducible DNA repair (SOS repair) appears to be involved in generating both types of mutational event following either UV- or γ-irradiation, at some stage in the processing of premutational lesions the level (or type) of DNA polymerase I activity in the cell seems to have an important role in determining whether or not frameshifts or base-pair substitutions will be produced at a particular frequency.

Effect of benzo[ a]pyrene on DNA synthesis and DNA polymerase activity of rat liver nuclei

When benzo[ a]pyrene (B[ a]P) was administered intraperitoneally to rats 48 hr before they were killed, the DNA-synthesizing capability of isolated rat liver nuclei was decreased as compared with control animals. B[ a]P also inhibited in vitro DNA synthesis in nuclei purified from control animals; this effect was enhanced by NADPH. DNA polymerases solubilized from purified nuclei of B[ a]P-treated animals were less active than those of control animals. DNA polymerase α was more inhibited than DNA polymerase β. Purified rat liver nuclei devoid of cytoplasmic contamination possess an NADPH-dependent B[ a]P hydroxylase activity. The observed inhibition of DNA synthesis in nuclei isolated from B[ a]P-treated rats was increased by NADPH. Moreover, there was an increased inhibition of DNA polymerase activity by nuclear membranes obtained from B[ a]P-treated animals when the incubations were performed in the presence of NADPH. Also, the derivative B[ a]P- trans-9,10-dihydrodiol was a potent inhibitor of DNA polymerase α under conditions where DNA polymerase β was less affected. These results suggest that nuclear B[ a]P hydroxylase might be involved in the inhibition of DNA synthesis probably at the level of DNA polymerase α. As in the in vivo studies, the nuclear polymerase most affected by the hydrocarbon in vitro was DNA polymerase α.

The DNA polymerase-primase from drosophila melanogaster embryos. Rate and fidelity of polymerization on single-stranded DNA templates.

The DNA polymerase activity of the near homogeneous, multisubunit DNA polymerase-primase from Drosophila melanogaster embryos has been compared to Escherichia coli DNA polymerase III core, DNA polymerase III, and DNA polymerase III holoenzyme. The rate of deoxynucleotide incorporation by the Drosophila polymerase on singly primed phi X174 DNA is similar to that observed with equivalent levels of DNA polymerase III holoenzyme in the absence of E. coli single-stranded DNA binding protein. However, analysis of the DNA products indicates that the Drosophila polymerase is less processive than DNA polymerase III holoenzyme, and closely resembles DNA polymerase III. The Drosophila polymerase-primase contains neither 3'-5' exonuclease nor RNase H-like activities, and catalyzes no significant pyrophosphate exchange. There is a low level of DNA-dependent ATPase activity which can be eliminated by a second glycerol gradient sedimentation (Kaguni, L.S., Rossignol, J.-M., Conaway, R.C., and Lehman, I.R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2221-2225). Although lacking a 3'-5' exonuclease, the replication fidelity of the D. melanogaster polymerase is similar to that of E. coli DNA polymerase III holoenzyme which possesses such an activity.

Plasmid cloning and expression of the E. coli polA + gene in S. cerevisiae

The E. coli polA (+) gene has been subcloned from a specialised λ transducing phage onto a low copy number plasmid. Plasmid-encoded DNA polymerase I was synthesised at 2 to 3 times the wild-type E. coli level, and was biochemically indistinguishable from chromosomally-encoded protein. It was able to counteract the radio sensitivity of polA1, polAex1, polAex2 and polA12 mutants, but no complementation of polA107 mutants occurred, even though the plasmid polA(+) gene was expressed. S. cerevisiae ars-1 or 2 μ replicative sequences were introduced into the polA(+) plasmid. Transformation of yeast with these constructs increased total DNA polymerase levels 2-20 times, depending upon assay conditions. The additional activity was discriminated from yeast DNA polymerases by its ability to use low concentrations of substrate, by its resistance to chemical inhibition, and by co-electrophoresis with pure DNA polymerase I and its proteolytic fragments. The polA(+) gene was expressed in yeast without the aid of yeast promotor sequences. However, deletion of cloned DNA more than 99 base pairs in front of the structural gene prevented expression in yeast but not in E. coli, indicating that the two organisms use different sequences for expression of the plasmid polA(+) gene.

Antiviral activity of the 3'-amino derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine.

3'-NH2-BV-dUrd, the 3'-amino derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, was found to be a potent and selective inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) replication. 3'-NH2-BV-dUrd was about 4-12 times less potent but equally selective in its anti-herpes activity as BV-dUrd. Akin to BV-dUrd, 3'-NH2-BV-dUrd was much less inhibitory to herpes simplex virus type 2 than type 1. It was totally inactive against a thymidine kinase-deficient mutant of HSV-1. The 5'-triphosphate of 3'-NH2-BV-dUrd (3'-NH2-BV-dUTP) was evaluated for its inhibitory effects on purified herpes viral and cellular DNA polymerases. Among the DNA polymerases tested, HSV-1 DNA polymerase and DNA polymerase alpha were the most sensitive to inhibition by 3'-NH2-BV-dUTP (Ki values 0.13 and 0.10 microM, respectively). The Km/Ki ratio for DNA polymerase alpha was 47, as compared with 4.6 for HSV-1 DNA polymerase. Thus, the selectivity of 3'-NH2-BV-dUrd as an anti-herpes agent cannot be ascribed to a discriminative effect of its 5'-triphosphate at the DNA polymerase level. This selectivity most probably resides at the thymidine kinase level. 3'-NH2-BV-dUrd would be phosphorylated preferentially by the HSV-1-induced thymidine kinase (Ki 1.9 microM, as compared with greater than 200 microM for the cellular thymidine kinase), and this preferential phosphorylation would confine the further action of the compound to the virus-infected cell.