DNA Polymerase Reaction Research Articles

Phosphorylation of nucleoside analogs by equine herpesvirus type 1 pyrimidine deoxyribonucleoside kinase

Replication of equine herpesvirus type 1 (EHV-1) was sensitive to 9-(1,3-dihydroxy-2-propoxymethyl)guanine(DHPG) but relatively resistant to E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). Likewise, plaque formation by EHV-1 was inhibited by DHPG, but not by BVDU. Plaque formation by a thymidine kinase-negative (tk-) mutant of EHV-1 was not inhibited by DHPG. In order to investigate biochemical mechanisms determining the differential sensitivity of EHV-1 to these drugs, the EHV-1-encoded thymidine kinase enzyme activity (TK)1 was partially purified from EHV-1-infected cells and analyzed. The EHV-1-induced enzyme utilized both ATP and CTP as phosphate donors and differed in relative electrophoretic mobility from the TKs of mock-infected and HSV-1-infected cells. Phosphorylation of 3H-dThd by the EHV-1 TK was inhibited by AraT, IdUrd, BVDU, and DHPG. The EHV-1 TK phosphorylated 125I-dCyd and 3H-ACV. The results indicate that EHV-1 encodes a pyrimidine deoxyribonucleoside kinase with broad nucleoside substrate specificity. These observations suggest that the failure of BVDU to inhibit EHV-1 replication is not attributable to an inability of the EHV-1 TK to phosphorylate BVDU, but may result from the incapacity of the viral TK to convert BVDU monophosphate to the triphosphate or from lack of inhibitory effect of BVDU triphosphate on viral DNA polymerase reactions.

Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells.

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.

Analysis of DNA polymerase reaction products for detecting hepatitis B virus in serum—comparison with spot hybridization technique

An assay for DNA polymerase reaction products using slab gel electrophoresis and autoradiography was compared with the spot hybridization technique for the detection of hepatitis B virus DNA in 317 blood samples. The former could identify the nature and size of DNA on electrophoresis, and reduce potentially false-positive results due to artifacts. Discordant results between the two methods occurred in 36 of 317 samples; 22 were positive by the spot technique alone, and 14 were positive by the analysis of DNA polymerase reaction products alone. However, the samples positive with the spot test alone showed weak radioactive signals on electrophoresis/autoradiography that were often interpreted as "inconclusive" by blind observations. Correlation of hepatitis B e antigen/antibody with hepatitis B virus DNA was studied in 91 patients with various chronic liver diseases. Discordant results, i.e., presence of the DNA in antibody positive sera, or its absence in the antigen positive sera, were obtained in 15 (19%) cases. Such patients tended to have advanced liver disease with fluctuating serum aminotransferase levels. Analysis of DNA polymerase reaction products by slab gel electrophoresis and autoradiography is not only sensitive, but is also as specific as the Southern blot technique in the detection of hepatitis B virus DNA in serum, and may prove useful in selected samples, especially where no cloned hepatitis B virus DNA is available, or in search of new hepatitis B virus-like viruses.

Maternal transmission of duck hepatitis B virus in pedigree Pekin ducks.

A 14-month old female Pekin duck experimentally infected as an embryo with duck hepatitis B virus via the amniotic route has been a chronic carrier of duck hepatitis B virus with very high (P/N) values of DNA polymerase activity since hatching. All the progeny were, on evaluation for congenital infection, found to be duck hepatitis B virus positive by endogenous DNA polymerase reaction and electron microscopy. These offspring remained persistently viremic throughout the study. Maternal transmission therefore bred true to a total of 49 offspring--24 ducklings (less than 24 hr old) and 25 ducks--studied. Six of these 25 ducks matched for age and sex and bled weekly for 6 weeks exhibited fluctuating plasma levels of DNA polymerase activity. Higher DNA polymerase activity was detected in newly hatched ducklings than in older viremic ducks. This observation was corroborated with the results of electron microscopic examination of thin sections of liver. Duck hepatitis B virus particles, located within vesicles of rough endoplasmic reticulum in the cytoplasm of hepatocytes, were more abundant, and therefore more readily observed, in ducklings than in older ducks.

Differential inhibition of DNA and RNA biosynthesis in HeLa S3 cells by tetaine, a dipeptide antibiotic

A dipeptide antibiotic, tetaine, was found to diminish the rate of incorporation of 3H-labelled precursors into nucleic acids of intact and permeabilized HeLa S3 cells with concomitant negligible effect on protein synthesis. Comparison of the inhibitory effects of tetaine indicates that the antibiotic at 0.03–0.1 mM is a selective inhibitor of cellular DNA biosynthesis and, at higher concentration, of DNA and RNA biosynthesis. Tetaine is also an inhibitor of DNA and RNA polymerase reactions in a cell-free system, as determined using partially purified extracts from HeLa S3 cells that served as a source of the enzymes. The pretreatment experiments showed that tetaine inactivated the polymerases without affecting DNA template function. The tetaine effect on biosynthesis of nucleic acids in HeLa S3 cells can be attributed rather to the intact antibiotic than to the product of its enzymatic cleavage, anticapsin.

Mouse DNA polymerase alpha-primase terminates and reinitiates DNA synthesis 2-14 nucleotides upstream of C2A1-2(C2-3/T2) sequences on a minute virus of mice DNA template.

The distribution of termination and initiation sites in a 5081-nucleotide minute virus of mice DNA template being copied by a highly purified mouse DNA polymerase alpha-DNA primase complex in the presence of GTP has been examined. The 3'-hydroxyl termini (17 in all) were clustered at six sites that were located 2-14 nucleotides upstream of C2A2C2, C2AC3, or C2A2T2 sequences. When either [alpha-32P]- or [gamma-32P]GTP was included in the DNA polymerase reaction mixtures, nascent DNA became radiolabeled. Analysis of the 32P-labeled material following treatment of the DNA with tobacco acid pyrophosphatase, bacterial alkaline phosphatase, or ribonuclease T1 revealed the presence of oligoribonucleotide chains averaging 5-7 nucleotides long and beginning with 5' GTP residues. Eight presumptive DNA primase initiation sites were located opposite C4 or C5 sequences 3-9 nucleotides upstream of one of the three closely related hexanucleotides C2A2C2, C2AC3, and C2A2T2. RNA-DNA junctions were found 3-10 nucleotides downstream of DNA primase initiation sites. The results indicate that hexanucleotides having the general formula C2A1-2(C2-3/T2), herein referred to as psi, are involved in promoting termination of DNA synthesis and/or de novo initiation of RNA-primed DNA chains by DNA polymerase alpha-primase.

Characterization of a Mr = 56,000 polypeptide associated with 10S DNA polymerase alpha purified from calf thymus using monoclonal antibody.

Existence of a Mr = 56,000 polypeptide associated with 10S DNA polymerase alpha was shown by production of a monoclonal anti-calf thymus 10S DNA polymerase alpha antibody secreted from a hybridoma line named 3H1. The antibody bound three polypeptides with Mr = 180,000, 56,000 and 32,000 in hydroxylapatite fraction of 10S DNA polymerase alpha by immunoblot. The antibody co-precipitated the polypeptides with the large polypeptide (Mr = 150,000-140,000) of 10S DNA polymerase alpha with the aid of second antibody. Among three polypeptides, the Mr = 56,000 polypeptide was co-purified with DNA polymerase alpha through DNA-cellulose chromatography and repeated sucrose rate-zonal centrifugations. The Mr = 56,000 polypeptide was still associated with 10S DNA polymerase alpha after second sucrose rate-zonal centrifugation, but the amount of it was reduced. The polypeptide was banded at pH 7.2-8.0 and displayed microheterogeneity in respect of isoelectric point by isoelectrofocusing with 7 M urea, and showed weak DNA-binding property after blotting onto a nitrocellulose. The antibody against the polypeptide precipitated DNA polymerase alpha from human, rat, and mouse, and Mr = 56,000 and 32,000 polypeptides were detected in these DNA polymerase alpha fractions by immunoblot. These results suggest that the polypeptide with Mr = 56,000 may take part in the DNA polymerase reaction.

Drug action on ribonucleotide reductase

Ribonucleotide reductase catalyzes the rate-limiting step in DNA synthesis. It represents a key metabolic site at which specific inhibitors have been directed as potential antitumor agents. Several different classes of ribonucleotide reductase inhibitors have been generated and studied. Because of the nature of the DNA polymerase reaction in which all four dNTPs are required, the initial velocity vs dNTP concentration curve gives sigmoidal rather than hyperbolic kinetics. As a result, a 50 per cent decrease in ribonucleotide reductase activity causes a decrease in DNA polymerase activity of 75 per cent or greater depending on the ratio of [dNTP] to its K m. This has been demonstrated with theoretical calculations, actual DNA polymerase determinations and precursor studies in intact tumor cells. The structural requirements for a compound to serve as a specific inhibitor of ribonucleotide reductase, either as the non-heme iron or effector-binding subunit, are stringent. Each protein subunit comprising the active enzyme can be specifically and independently inhibited. When combinations of agents, each directed at one of the subunits of ribonucleotide reductase, are used, strong synergistic inhibition of L1210 cell growth and synergistic cytotoxicity result.

Interaction with nucleic acids and stimulation of the viral DNA polymerase by the herpes simplex virus type 1 major DNA-binding protein

The interaction of the herpes simplex virus type 1 (HSV-1) major DNA-binding protein, infected-cell polypeptide 8 (ICP8), with nucleic acids has been examined by a filter-binding assay and electron microscopy. Filter-binding assays done over a broad pH range indicated that the optimum pH for the protein-DNA interaction is approximately 7.6. Heat inactivation studies showed that ICP8 is stable at temperatures up to 40 degrees C with a rapid loss of binding activity on incubation at 45 degrees C and above. Competition binding experiments have established the following relative affinities of ICP8 for the following nucleic acids: single-stranded HSV-1 DNA congruent to bacteriophage fd DNA greater than polyriboadenylate much greater than double-stranded HSV-1 DNA congruent to d(pCpT)5. Observation of negatively stained ICP8-single-stranded DNA complexes indicated that ICP8 binds along the length of the DNA in a regular repeating fashion. The average width of these complexes is 9.3 +/- 0.8 nms. Finally. Finally, addition of purified ICP8 to HSV-1 DNA polymerase reactions resulted in a stimulation of the viral polymerase activity.

Hepatitis B viral DNA-RNA hybrid molecules in particles from infected liver are converted to viral DNA molecules during an endogenous dna polymerase reaction

Particles purified from the liver of hepatitis B virus-infected patients were previously shown by us to incorporate 32P-deoxynucleotides into viral DNA and DNA-RNA hybrid molecules when incubated in a DNA polymerase reaction mixture. In this investigation, similar particles from duck and ground squirrel livers infected with viruses closely related to HBV were also shown to incorporate 32P-deoxynucleotides into viral-specific DNA and DNA-RNA hybrid molecules when incubated in vitro in a DNA polymerase reaction mixture. The particles from duck hepatitis B virus-infected liver contained a heterogeneous population of hybrid molecules, while those from ground squirrel hepatitis virus-infected liver contained hybrid molecules with densities similar to those found in HBV particles including a distinct population of molecules with an average density of 1.57 g/cm 3. Brief endogenous DNA polymerase reactions with particles from all three livers, resulted in incorporation of 32P-deoxynucleotides into viral DNA of DNA-RNA hybrid as well as viral DNA molecules. When the reaction was continued in the presence of a 1000-fold molar excess of unlabeled deoxynucleotides, a decrease in [ 32P]DNA in the DNA-RNA hybrid region of the Cs 2SO 4 density gradient and a proportional increase in [ 32P]DNA in the DNA region of the gradient was observed. This effect was seen most dramatically with particles isolated from freshly obtained ground squirrel hepatitis virus-infected livers in which 90% of the pulse labeled DNA in the hybrid species at the buoyant density of 1.57 g/cm 3 appeared to be converted to a form with the buoyant density of pure DNA (1.42 g/cm 3). Storage of virus particles at 4°, or prior freezing of infected ground squirrel liver almost completely abolished the ability of the endogenous DNA polymerase activity to incorporate 32P-deoxynucleotides into hybrid molecules, while incorporation into DNA molecules was apparently unaffected. These results suggest that different enzymatic activities catalyze synthesis of the viral DNA in DNA-RNA hybrids and in molecules with buoyant density of pure DNA. Thus particles from infected liver synthesize DNA of DNA-RNA hybrid molecules which can be converted in the particles into molecules with the buoyant density of pure DNA. This indicates that DNA-RNA hybrids may be intermediates in viral DNA replication and that the mechanism of hepatitis B virus (and closely related viruses of ground squirrels and ducks) DNA replication differs from that known for other DNA viruses.

Hepatitis B virus particles of plasma and liver contain viral DNA-RNA hybrid molecules

Hepatitis B virions in plasma (Dane particles) are known to contain small circular DNA molecules. The experiments described here indicate that virions in plasma, as well as particles from hepatitis B virus-infected human liver, also contain viral DNA-RNA hybrid molecules, and deoxynucleotides can be incorporated into the DNA of these hybrids by DNA polymerase activities in the virions. Thus, two viral DNA synthetic reactions appear to take place in virions: repair of the single-stranded region of circular DNA molecules and synthesis or elongation of the DNA strand of DNA-RNA hybrid molecules. Centrifugation of virion nucleic acid to equilibrium in Cs 2SO 4 density gradients revealed the presence of viral DNA-RNA hybrid molecules over a density range of 1.45 to 1.60 g/cm 3. Distinct species of hybrid molecules were found with an average density of 1.57 g/cm 3 in Dane particles and 1.52 and 1.57 g/cm 3 in particles from liver. Fractionation of nucleic acid from Cs 2SO 4 density gradients by gel electrophoresis demonstrated that the majority of hybrid molecules migrated faster than molecules with the density of pure DNA (1.42 g/cm 3). One notable exception was the finding of DNA-RNA hybrid molecules migrating slower than open circular viral DNA. Characterization of viral DNA-RNA hybrids by heat denaturation Cs 2SO 4 density gradient fractionation, and recombinant ] single-stranded probe hybridization revealed that the hybrid molecules consisted of viral plus-strand RNA hydrogen bonded to viral minus-strand DNA sequences. Data obtained by pancreatic ribonuclease digestion revealed that the hybrid molecules at density 1.45 to 1.52 g/cm 3 contained HBV RNA strands base paired over only part of their length in contrast to the hybrid species at density 1.57 g/cm 3 which contained RNA strands apparently base paired over most of their length. Further characterization showed that the hyrid at 1.57 g/cm 3 contained genome-length minus-strand viral DNA. The experiments rule out the possibility that the hybrid molecules are transcriptional complexes. Data presented in a companion manuscript indicate that the hybrid molecules may represent intermediates in the synthesis of viral DNA in the endogenous DNA polymerase reaction.

Hepatitis B virus DNA forms in nuclear and cytoplasmic fractions of infected human liver

Human liver tissues obtained at autopsy from two patients chronically infected with hepatitis B virus (HBV) were found to contain several distinct species of HBV DNA. Southern blot analysis using a nick-translated HBV [ 32P]DNA probe identified specific DNA bands migrating at the positions expected for linear double-stranded DNA of 3.6 and 2.0 kb. These DNA bands were shown to represent relaxed circular and closed circular (supercoiled) HBV DNA, respectively. In addition to these distinct bands several minor bands as well as a heterogeneous population of HBV DNA molecules were present. When infected cell nuclei were isolated, and the nuclear and cytoplasmic nucleic acid separately analyzed, the nuclear fraction contained the 2.0-kb DNA species. This species was shown to be supercoiled 3.2-kb HBV DNA by electron microscopy, restriction endonuclease digestion, and thermal denaturation. The cytoplasmic fraction contained DNA forms similar to those found in virions isolated from plasma (i.e., migration in the position of linear double-stranded molecules of 3.6 and 3.2 kb) and no supercoiled DNA was detected. Particles isolated from the cytoplasmic fraction were able to incorporate dNTPs into viral DNA sequences. Southern blot analysis of the nucleic acid isolated from the particles revealed the presence of HBV DNA forms migrating in positions expected for 3.6- and 3.2-kb linear double-stranded molecules as well as a heterogeneous population of HBV molecules. The 3.6- and 3.2-kb species were identified as relaxed circular and double-stranded linear genome-length HBV DNA. Digestion of the viral nucleic acid with pancreatic ribonuclease increased the electrophoretic mobility of a portion of the heterogeneous HBV molecules and resulted in the appearance of a distinct 1.9-kb DNA band suggesting the same viral DNA was complexed with RNA. Experiments to be reported elsewhere showed this DNA species to be genome-length minus-strand HBV DNA which was released from DNA-RNA hybrid molecules by RNase digestion. Thus, supercoiled HBV DNA exists free in the nucleus of infected liver cells and cytoplasmic particles contain relaxed circular and linear HBV DNA as well as a heterogeneous population of HBV DNA and DNA-RNA hybrid molecules, and a DNA polymerase reaction in the particles results in incorporation of dNTP into DNA strands of these molecules.

Inhibition of human and woodchuck hepatitis virus DNA polymerase by the triphosphates of acyclovir, 1-(2′-deoxy-2′-fluoro-β- d-arabinofuranosyl)-5-iodocytosine and E-5-(2-bromovinyl)-2′-deoxyuridine

The triphosphates of acyclovir (ACV), 1-(2′-deoxy-2′-fluoro-β- d-arabinofuranosyl)-5-iodocytosine (FIAC) and E-5-(2-bromovinyl)-2′-deoxyuridine (BVdU) have been examined for their inhibitory effects on the endogenous DNA polymerase reactions of human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV). All three triphosphates (ACVTP, FIACTP and BVdUTP) inhibited the HBV and WHV DNA polymerases by competing with the corresponding natural substrates. FIACTP was the most potent inhibitor of HBV and WHV DNA polymerase while ACVTP was the least effective inhibitor. The inhibitory properties of these compounds were compared with those of the 5′-triphosphates of 1-β-arabinofuranosylcytosine (ara-CTP) and 1-β-arabinofuranosylthymine (ara-TTP). The 50% inhibitory doses for HBV and WHV DNA polymerases were in the following order: FIACTP < BVdUTP < ara-TTP < ACVTP < ara-CTP. BVdUTP appeared to be an efficient alternate substrate to dTTP for HBV DNA polymerase while FIACTP was much less efficient when substituted for dCTP. ACVTP did not act as an alternate substrate to dGTP and appeared to prevent DNA chain elongation.

Presence of retrovirus in the B95-8 Epstein-Barr virus-producing cell line from different sources.

The B95-8 cell line, a widely used source of highly transforming Epstein-Barr virus (EBV), obtained from the laboratory of origin, harbored an infectious retrovirus. This retrovirus generally resembled the Type D retroviruses structurally and developmentally and like the Type D retroviruses preferred Mg2+ to Mn2+ in its RNA-directed DNA polymerase reaction. Evidence for the presence of retrovirus was found in B95-8 cultures from two other sources within the United States, either by assay for polymerase or by electron microscopy. Comparison of two B95-8 cell lines showed cytogenetic differences as well as differences in retroviral activities. The results suggest that any B95-8 culture should be tested for the presence of retrovirus before its use as a source of EBV.

Single mutations at many sites within the DNA polymerase locus of herpes simplex viruses can confer hypersensitivity to aphidicolin and resistance to phosphonoacetic acid.

Aphidicolin, a tetracyclic diterpenoid which inhibits the DNA polymerase-alpha activities of many eukaryotic cells, inhibited herpes simplex virus growth and DNA synthesis in infected cultures and the activity of the virus DNA polymerase in vitro. A wide range of stable aphidicolin sensitivities was represented amongst a collection of virus strains with no prior exposure to this drug, but viruses with polymerase mutations selected for resistance to phosphonoacetic acid (PAA) or to acycloguanosine typically showed increased sensitivity to aphidicolin. Of 16 unrelated PAA-resistant variants, 7 were hypersensitive to aphidicolin. A number of mutants with temperature-sensitive (ts) lesions in the polymerase gene also showed increased aphidicolin sensitivity (e.g. HSV-1[mP17]tsH) or aphidicolin hypersensitivity (e.g. HSV-1[KOS]tsD9, tsC4). Resistance or hypersensitivity of virus growth and DNA synthesis in vivo were correlated with resistance or hypersensitivity of virus DNA polymerase reactions in vitro. Resistance phenotypes were closely linked to the polymerase gene during recombination with outside markers. Moreover, the selection of aphidicolin-resistant mutants from hypersensitive variants with independent PAA resistance or ts mutations in the polymerase gene could result in co-selection for PAA-sensitive and ts+ phenotypes. Confirmation that multiple independent mutations could determine aphidicolin hypersensitivity was obtained by studies of recombination between independent hypersensitive variants. Aphidicolin-resistant recombinant progeny were formed with recombination frequencies (0.4 to 2.6%) compatible with intragenic events. With parental hypersensitive variants which were products of limited PAA selection, or with the ts polymerase mutations, aphidicolin-resistant recombinants were PAA-sensitive and/or ts+. The segregation of other markers (ts, plaque morphology) amongst recombinant progeny permitted the orientation of multiple determinants of PAA resistance and aphidicolin hypersensitivity with respect to other markers in the polymerase gene and in other genes. The nature of residues determined at any one of a constellation of separate sites within the polymerase locus can determine resistance or sensitivity to antiviral drugs and aphidicolin hypersensitivity associated with changes at the polymerase locus facilitates high resolution genetic analysis of this locus.

Interaction of new anthracycline antibiotics with DNA. Effects on nucleic acid synthesis and binding to DNA

The effects of various new anthracycline antibiotics on DNA and RNA synthesis were studied using DNA polymerase I (EC 2.7.7.7), RNA polymerase (EC 2.7.7.6) obtained from Escherichia coli and reverse transcriptase obtained from avian myeloblastosis virus. Aclacinomycin A, its analogues, baumycins A1 and A2, adriamycin and daunomycin showed potent inhibitory effects on these polymerases, with calf thymus DNA as template, with IC 50 values of 10–30 μM. With poly(rA)·d(pT) 10 as template for reverse transcriptase, aclacinomycin A and daunomycin showed IC 50 values higher than 500 μM. Baumycins B1, B2, C1, and C2 showed high IC 50 values on three polymerases. Addition of excess template DNA to the reaction mixture reversed the inhibitory effect of anthracyclines. Addition of calf thymus DNA to anthracyclines caused a bathochromic and hypochromic change in the visible spectrum. Apparent binding constant for aclacinomycin A, its analogues, and adriamycin were in the range of (1–2)·10 6M −1. Aclacinomycin A and adriamycin also bind to heat denatured DNA, but not strongly to yeast RNA. From these results, the structure-activity relationships of new anthracyclines on DNA binding and polymerase reactions are discussed.

The mode of action of cytotoxic and antitumor 1-nitroacridines. I. The 1-nitroacridines do not exert their cytotoxic effects by physicochemical binding with DNA

The mode of action of cytotoxic and antitumor 1-nitroacridines and their isomeric derivatives was studied by comparing their effects in cell-free systems and towards cultured tumor HeLa cells, assuming that the nitroacridines considered exert cytotoxic effects by physicochemical binding with the DNA. All the nitroacridines impaired biosyntheses of DNA, RNA and protein in cultured HeLa cells and a causal relationship between nitroacridine inhibition of macromolecular biosyntheses and lethal effects of the agents appears likely. In cell-free systems, the nitroacridines bound with two independent sites on the DNA, forming complexes with enhanced resistance to DNA strand separation upon melting and inhibited the DNA polymerase reaction by altering activity of template and/or of enzyme. The 1-nitroacridines were poorly effective in cell-free systems and were the most potent inhibitors toward the growth of HeLa cells among the derivatives studied. It is concluded that the primary events responsible for cytotoxic effects of antitumor 1-nitroacridines and of their isomeric derivatives are different. The metabolic activation of 1-nitroacridines to more reactive intermediates which will attach to and alter the structure and/or function of DNA of sensitive cells is suggested.

Studies on catalytic subunits of mouse myeloma alpha-polymerase.

Activity gel analysis has been used to identify mouse myeloma polypeptides with DNA polymerase activity. Proteins in a crude homogenate of mouse myeloma were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sodium dodecyl sulfate was soaked from the gel, and polypeptides were allowed to renature in situ; then the intact gel was incubated in a DNA polymerase reaction mixture in order to localize DNA polymerases (Spanos, A., Sedgwick, S. G., Yarranton, G. T., Hübscher, U., and Banks, G. R. (1981) Nucleic Acids Res. 9, 1825-1839). This activity gel analysis revealed that the homogenate contains a Mr = 40,000 polypeptide with strong DNA polymerase activity; from its Mr and catalytic properties this enzyme was identified as beta-polymerase. The homogenate also contains two additional DNA polymerase activities giving relatively strong bands at Mr = approximately 76,000 and approximately 120,000, respectively. Results on the catalytic properties of both of these enzymes suggest that they are alpha-polymerases. Further evidence for the existence of a Mr approximately 120,000 alpha-polymerase catalytic polypeptide came from the observation that a purified preparation of one of the recognized species of mouse myeloma alpha-polymerase is composed of a Mr = approximately 120,000 polypeptide, as revealed by Coomassie blue staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 3 M urea. This purified enzyme does not contain polypeptides in the Mr = 40,000 to 70,000 range, and is capable of producing a strong band at Mr = 120,000 in the activity gel assay.

Terminal deoxynucleotidyl transferase help DNA polymerase alpha bypass thymine dimer.

The physiological function of terminal deoxynucleotidyl transferase (EC 2.7.7.31) is not known. In 1974, Baltimore (1) proposed a model in which terminal transferase acts as a somatic mutator at a special region of immunoglobulin gene during the maturation of immunocompetent cells. On the other hand, a speculative mechanism for induced mutagenesis in the eukaryotic cell was also discussed in connection with the possible induction of terminal transferase in the cell which has damaged DNA (2), In order to verify these hypothesis, further biochemical analysis may be required with respect to a possible modulation of the DNA polymerase reaction by terminal transferase. Recently, we have shown (3) that the replication by DNA polymerase α stops at the site of thymine dimer induced by ultraviolet-irradiation on the template poly(dT). The addition of terminal deoxynucleotidyl transferase to this system enhanced the DNA synthesis to the control level and it concemittantly increased the incorporation of the mismatched deoxynucleotides into the newly synthesized poly(dA) strand. The sizes of newly synthesized DNA were smaller with ultraviolet irradiated template but they increased to the control level with the addition of terminal deoxynucleotidyl transferase to the system. These results suggest that terminal deoxynucleotidyl transferase can help DNA polymerase α “bypass” thymine dimers in vitro by the formation of mismatched regions at the positions opposite to pyrimidine dimers on the template.

Inhibition of purified human and herpes simplex virus-induced DNA polymerases by 9-(2-hydroxyethoxymethyl)guanine triphosphate. Effects on primer-template function.

The inhibition of highly purified herpes simplex virus (HSV)-induced and host cell DNA polymerases by the triphosphate form of 9-(2-hydroxyethoxymethyl)guanine (acyclovir; acycloguanosine) was examined. Acyclovir triphosphate (acyclo-GTP) competitively inhibited the incorporation of dGMP into DNA, catalyzed by HSV DNA polymerase; apparent Km and Ki values of dGTP and acyclo-GTP were 0.15 microM and 0.003 microM, respectively. HeLa DNA polymerase alpha was also competitively inhibited; Km and Ki values of dGTP and acyclo-GTP were 1.2 microM and 0.18 microM, respectively. In contrast, HeLa DNA polymerase beta was insensitive to the analogue. The "limited" DNA synthesis observed when dGTP was omitted from HSV or alpha DNA polymerase reactions was inhibited by acyclo-GTP in a concentration-dependent manner. Prior incubation of activated DNA, acyclo-GTP, and DNA polymerase (alpha or HSV resulted in a marked decrease in the utilization of the primer-template in subsequent DNA polymerase reactions. This decreased ability of preincubated primer-templates to support DNA synthesis was dependent on acyclo-GTP, enzyme concentration, and the time of prior incubation. Acyclo-GMP-terminated DNA was found to inhibit HSV DNA polymerase-catalyzed DNA synthesis. Kinetic experiments with variable concentrations of activated DNA and fixed concentrations of acyclo-GMP-terminated DNA revealed a noncompetitive inhibition of HSV-1 DNA polymerase. The apparent Km of 3'-hydroxyl termini was 1.1 X 10(-7) M, the Kii and Kis of acyclo-GMP termini in activated DNA were 8.8 X 10(-8) M and 2.1 X 10(-9) M, respectively. Finally, 14C-labeled acyclo-GMP residues incorporated into activated DNA by HSV-1 DNA polymerase could not be excised by the polymerase-associated 3',5'-exonuclease activity.