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We have examined the capacity of a series of 6-substituted pyrimidine and 2-substituted purine derivatives to inhibit mammalian thymidine kinase and the thymidine kinases encoded by type 1 and type 2 herpes simplex viruses. Several N2-substituted guanine and deoxy guanosine derivatives displayed selective inhibitory activity against the HSV-1 and HSV-2 thymidine kinases by competing with the phosphorylation of thymidine, suggesting a possible novel pharmacological approach to herpes viruses infections.

Aphidicolin inhibits DNA replication and growth of all tested human and murine neoplastic cells including leukemic T- and B-lymphocytes and melanocarcinoma cells. The concentration of aphidicolin causing 50% inhibition of DNA synthesis in all of the tested neoplastic cell lines is similar to that necessary to inhibit DNA synthesis in HeLa cells by 50%. The mechanism of inhibition of DNA synthesis in neoplastic cells is again due to the inhibition of DNA polymerase alpha by aphidicolin. Aphidicolin at a concentration 100 times higher than that causing 50% inhibition of DNA synthesis and cell growth had no effect on total protein synthesis, on the secretion of immunoglobulins, or on the expression of HLA antigens which are involved in relevant phenomena of the immune response.

Studies of a variety of compounds designed as derivatives of prototype active molecules aphidicolin and doxorubicin are reported. So far none of the aphidicolin simpler analogues is as active as the parental molecule. Ten anthracycline analogues, characterized for their cytotoxicity, antitumor activity and inhibition of the relaxing activity of purified human DNA topoisomerase II can be divided into five groups. The majority of the tested compounds shows properties very similar to those of doxorubicin. Epirubicin shows extremely high inhibitory activity toward the relaxing property of topoisomerase II but its antitumor activity and cytotoxicity are similar to those of the former group. The third group includes a compound with extremely high cytotoxicity. The fourth group is represented by a compound which shows a cytotoxicity. The fourth group is represented by a compound which shows a cytotoxicity. The fourth group is represented by a compound which shows a cytotoxicity typical of anthracyclines and good antitumor activity but which has no specific inhibitory activity on topoisomerase II. A fifth group includes a totally inactive compound. Our results suggest that the inhibition of human DNA topoisomerase II is only partially correlated with antitumor activity.

Aphidicolin, a tetracyclic diterpenoid obtained from the culture filtrates of Cephalosporium aphidicola and other fungi, inhibits the growth of eukaryotic cells and of certain animal viruses (SV40, Herpes and Vaccinia viruses) by selectively inhibiting the cellular replicative DNA polymerase alpha or the viral-induced DNA polymerases. The arrest of cellular or viral growth is thus due to inhibition of cellular or viral replicative DNA synthesis without interference with mitochondrial DNA synthesis, RNA, protein and nucleic acid precursors synthesis or other major metabolic pathways. The inhibition of all sensitive eukaryotic DNA polymerases by aphidicolin is competitive with respect to dCTP. Aphidicolin has thus proved extremely useful in elucidating the functional role of DNA polymerase alpha in nuclear DNA replication, of DNA polymerase gamma in mitochondrial DNA synthesis and both DNA polymerases beta and alpha in DNA repair synthesis. An important laboratory application of aphidicolin is the synchronization of the cell cycle of eukaryotic cells both in culture and in vivo. The properties of aphidicolin have recently aroused considerable interest for its possible exploitation in al practice. The mechanism of action of this drug suggests in fact that it may be useful for controlling excessive cell proliferation in patients with cancer, psoriasis or other dermatitis with little or no adverse effect upon non-multiplying cells. Interestingly, when administered to mice, the highest levels of aphidicolin are found in those tissues most actively proliferating with little or no aphidicolin present in neurons or myocardial cells.

We have established a novel procedure to purify calf thymus DNA polymerase delta from cytoplasmic extracts. The enzyme has typical properties of DNA polymerase delta including a 3' - greater than 5' exonuclease activity and efficiently replicates natural occurring genomes such as primed single-stranded M13 DNA and single-stranded porcine circovirus DNA, this last one thanks to an associated or contaminating primase activity. A processivity of at least a thousand bases was evident and this in the apparent absence of proliferating cell nuclear antigen. The enzyme was purified through a procedure that allows the simultaneous isolation of DNA polymerase delta, DNA polymerase alpha-primase and a DNA dependent ATPase. All these enzymes coeluted from a phosphocellulose column. After chromatography on hydroxylapatite DNA polymerase delta separated from the coeluting DNA polymerase alpha and DNA dependent ATPase. Separation of the latter two was achieved on heparin-Sepharose. DNA polymerase delta was further purified by heparin-Sepharose and fast protein liquid chromatography. Purified DNA polymerase delta was resistant to the DNA polymerase alpha inhibitors BuPdGTP and BuAdATP and did not react with DNA polymerase alpha monoclonal and polyclonal antibodies. Based on this isolation protocol we can start to test biochemically the hypothesis whether DNA polymerase delta and DNA polymerase alpha might act coordinately at the replication fork as leading and lagging strand replicases, respectively.

We have developed a method, based on the in vitro inhibition of purified human DNA polymerase alpha, the major enzyme of DNA replication, which allows the rapid and accurate determination of pmol amounts of aphidicolin, a promising anticancer drug. The efficacy of this simple method was verified by the determination of aphidicolin in the liver, spleen, blood and urine of mice treated parenterically with the drug. Given its sensitivity and the avoidance of radioactive tracers, this enzymatic method is suitable for the determination of the drug in body fluids and tissue biopsies from living humans. It allows the detection and quantitation of aphidicolin in the presence of inactive metabolite(s) with very similar chemical structure(s) such as those generated by liver microsomal oxidases. The technique will also be useful to monitor the purification of the drug from cultures of Cephalosporium aphidicola.

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64-66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39-57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase alpha and delta as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase alpha and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase alpha holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789-4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase alpha is blocked with the DNA polymerase alpha specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase delta can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.

DNA polymerase delta from calf thymus was purified under conditions that minimized proteolysis to a specific activity of 27,000 units/mg. The four step isolation procedure included phosphocellulose, hydroxyapatite, heparin-Sepharose and FPLC-MonoS. This enzyme consists of four polypeptides with Mr of 140, 125, 48 and 40 kilodaltons. Velocity gradient sedimentation in glycerol removed the 48 kDa polypeptide while the other three sedimented with the DNA polymerase activity. The biochemical properties of the three subunit enzyme and the copurification of 3'----5' exonuclease activity were typical for a bona fide DNA polymerase delta. Tryptic peptide analysis showed that the 140 kDa polypeptide was different from the catalytic 180 kDa polypeptide of calf thymus DNA polymerase alpha. Both high Mr polypeptides (140 and 125 kDa) were catalytically active as analysed in an activity gel. Four templates were used by DNA polymerase delta with different preferences, namely poly(dA)/oligo(dT)12-18 much much greater than activated DNA greater than poly(dA-dT) greater than primed single-stranded M13DNA. Calf thymus proliferating cell nuclear antigen (PCNA) could not stimulated this DNA polymerase delta in any step of the isolation procedure. If tested on poly(dA)/oligo(dT)12-18 (base ratio 10:1), PCNA had no stimulatory effect on DNA polymerase delta when tested with low enzyme DNA ratio nor did it change the kinetic behaviour of the enzyme. DNA polymerase delta itself did not contain PCNA. The enzyme had an intrinsic processivity of several thousand bases, when tested either on the homopolymer poly(dA)/oligo(dT)12-18 (base ratio 64:1) or on primed single-stranded M13DNA. Contrary to DNA polymerase alpha, no pausing sites were seen with DNA polymerase delta. Under optimal in vitro replication conditions the enzyme could convert primed single-stranded circular M13 DNA of 7,200 bases to its double-stranded form in less than 10 min. This supports that a PCNA independent DNA polymerase delta exists in calf thymus in addition to a PCNA dependent enzyme (Lee, M.Y.W.T. et al. (1984) Biochemistry 23, 1906-1913).

The activity ratio of DNA polymerases delta and alpha in calf thymus was found to be invariably 1:1, irrespective of extraction procedure (8 types) and subcellular localization (cytoplasm, nucleus and microsomes). This was established by separation of the two forms by hydroxyapatite chromatography and by their response to specific inhibitors and monoclonal antibodies. This finding supports the dimeric DNA polymerase model [(1980) J. Biol. Chem. 255, 4290-4303], which proposes that DNA polymerases delta and alpha act coordinately as leading and lagging strand enzymes, respectively, at the replication fork.

Parameters defining the topological state of DNA seem extremely important for describing the reactive state of the same DNA molecules. We have shown that physical and chemical DNA modifying agents alter the tertiary structure of DNA molecules. Variations in the tertiary structure of DNA were studied by one dimensional electrophoresis on an agarose gel of partially relaxed plasmid DNA topoisomers, a technique allowing the measurement of alterations in the degree of supercoiling equivalent to fractions of superhelical turns. Unwinding angles of -10.1 degrees or -8.7 degrees per pyrimidine or thymine dimer respectively, of -12 degrees per apurinic site, and of -3.4 degrees per methylated purine were obtained by titrating the number of damaged sites necessary to reduce the number of superhelical turns by one in each topoisomer. On the contrary, enzymatic methylation of the C-5 position of cytosine (a modified base present in prokaryotic and eukaryotic DNAs) did not alter the DNA tertiary structure. We have also shown that local alterations in DNA structure caused by UV-irradiation inhibit bacterial DNA topoisomerase I and DNA methylase, and that the topological state of DNA substrate influences the mode of methylation of Hpa II DNA methylase. These findings suggest that the natural topological state of DNA substrate (linear, relaxed, or covalently closed duplex DNA with varying degrees of supercoiling) influences the mode of action of enzymes possibly involved in DNA repair processes, while DNA structural alterations caused by DNA modifying agents might influence DNA repair processes in two ways: either by driving the interaction between repair enzymes and the modified sites of DNA, or by inhibiting or changing the mode of action of enzymes normally acting on unmodified DNA.

A series of N2-substituted guanine derivatives was screened against mammalian thymidine kinase and the thymidine kinase encoded by type I herpes simplex virus to examine their capacity to selectivity inhibit the viral enzyme. Several bases, nucleosides, and nucleotides displayed selective activity. The mechanism of action of the most potent derivative, N2-phenyl-2'-deoxyguanosine (PhdG) was studied in detail. PhdG (a) inhibited the viral enzyme competitively with respect to the substrates thymidine and deoxycytidine, (b) was completely resistant to phosphorylation, (c) displayed limited toxicity for the HeLa cell lines employed as hosts for viral infection, and (d) selectively inhibited viral thymidine kinase function in intact cultured cells. The results indicate that the PhdG drug prototype has potential as a selective anti-herpes agent and as a novel molecular probe of the structure and function of herpes simplex thymidine kinase.

DNA polymerase delta, a fourth cellular DNA polymerase, might play an important role in cellular events. The properties of the enzyme suggested possible roles in nuclear DNA replication and DNA repair. By using specific assays that allow the determination of DNA polymerases delta and alpha in crude enzyme fractions (1), we show here that in developing rat neurons, which stop dividing before birth, both DNA polymerases delta and alpha drop sharply in an identical pattern from a high level with the approach of term and disappear at approximately three weeks of postnatal age. The results suggest that DNA polymerases delta and alpha might have a coordinated function during DNA replication in eukaryotic cells.