No abstract

By equilibrium dialysis a diadenosine 5',5"'-P',P2-tetraphosphate (Ap4A) This enzyme was applied on a 15 X 1.5 cm column of native calf thymus DNA-cellulose. The column was washed with' 200 ml of 20 mM Tris1HCI, pH 8.0/7 mM 2-mercaptoethanol/20% glycerol and the enzyme was'eluted with a 500-ml salt gradient (0-200 mM potassium chloride). The' polymerase a-containing fractions were concentrated by vacuum dialysis and further purified by gel filtration. The enzyme solution (4 ml) was applied qn a Bjo-Gel A 0.5 column Abbreviation: Ap4A, diadenosine 5',5"-PlP4-tetraphosphate.

The physiological functions of DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) (3 and y were investigated by using neuronal nuclei and synaptosomes isolated from rat brain. UV irradiation of neuronal nuclei from 60-day-old rats resulted in a 7-to 10-fold stimulation of DNA repair synthesis attributable to DNA polymerase # which, at this developmental stage, is virtually the only DNA polymerase present in the nuclei. No repair synthesis could be elicited by treating the nuclei with N-methyl-N-nitrosourea, but this was probably due to the inability of brain tissue to excise alkylated bases from DNA. The role of DNA polymerase y was studied in synaptosomes by using a system mimicking in vivo mitochondrial DNA synthesis. By showing that, under these conditions, DNA The direct assignment of functions to the three mammalian DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) (a, (, and y) in DNA replication and repair is hampered by the lack of conditional mutants defective in DNA synthesis. To date, one is forced to admit that the evidence presented is at best circumstantial. Despite these reservations, some progress has been made concerning the function of DNA polymerase a. A wealth of information indicates that this enzyme has a major role in DNA replication (reviewed in refs. 1 and 2). Recently, this idea has been further strengthened by the finding that DNA polymerase (v is the major polymerase associated with replicating simian virus 40 chromosomes (3-5). In addition, all aspects of in vitro replicative synthesis by these chromosomes (3) or HeLa cell lysates (6) were found to be resistant to 2',3'-dideoxythymidine 5'-triphosphate (d2TTP), a specific inhibitor of DNA polymerases ( and By but not of DNA polymerase a (3, 6). Conversely, aphidicolin, a selective inhibitor of DNA polymerase at, prevents mitotic division of sea urchin embryos, which requires replicative DNA synthesis, but it has no effect on meiotic division in starfish oocytes, which is not dependent on DNA replication (7). Taken together these observations strongly indicate that DNA polymerase ae is the nuclear replicating enzyme.In contrast, the physiological functions of DNA polymerases (14) and synaptosomea (15) were prepared from the forebrain cortex of 60-day-old and 14-day-old rats (SIV-50 strain), respectively. The preparations were frozen immediately at -70'C until use. Nuclei were counted by using a flow cytophotometer (Phywe, Gottingen, Germany), and protein concentrations were determined by the method of Lowry et al. (16). Repair DNA synthesis in neuronal nuclei and synaptosomes was assayed as described in Table 1.In Vitro System Supporting mtDNA Synthesis in Synaptosomes. Synaptosomes were suspended in 0.32 M sucrose/1 mM KH2PO4, pH 7.5/0.1 mM EDTA and were made permeAbbreviations: BrdUTP, 5-bromo-2'-deoxyuridine 5'-triphosphate; d2TTP, 2',3'-dideoxythymidine 5'-triphosphate; MNU,KN-methyl-N-nitrosourea. t Present address:

DNA polymerase p was isolated from rat cortex neurons and characterised. Its properties were strikingly similar to those of other mammalian /?-polymerases. In adult rats, this was the major DNA polymerase occurring in neuronal nuclei, which contained no a-polymerase, 99.2 % /?-polymerase and only 0.8 % y-polymerase. Isolated neuronal nuclei of this developmental stage were shown to perform ultraviolet-induced repair DNA synthesis in virro. Since /?-polymerase was virtually the exclusive DNA polymerase in these nuclei it was concluded that the p enzyme was responsible for the observed DNA repair. This was further substantiated by demonstrating a virtually complete suppression of DNA repair in irradiated nuclei by 2',3'-dideoxyribosylthymine 5'-triphosphate (d2TTP), a potent /?-polymerase inhibitor. However, the presence of minute amounts of y-polymerase in neuronal nuclei and its susceptibility to d2TTP did not allow one to rule out an ancillary role of DNA polymerase y in DNA repair. In view of the similarity of the neuronal DNA polymerase /? with all other mammalian /?-polymerases it may be speculated that the ability to perform repair DNA synthesis is not unique to the neuronal enzyme but is a general function of all /?-polymerases.Our interest in the neuronal DNA polymerases derives from the observation that cell-cycle-independent replicative DNA synthesis occurs in cortical neurons during early postnatal development resulting in a permanent DNA increase [l -41. We have therefore initiated a survey of the enzymology of neuronal DNA synthesis and, in particular, of the DNA polymerases M, p and y [3,5-81 (for recent reviews on the three mammalian DNA polymerases, see 19 -111). As a first approach we have analysed the mitochondria1 DNA polymerase of synaptic terminals and have shown this to be identical with DNA polymerase y from brain nuclei [6]. Subsequently, we have identified the mitochondrial DNA polymerase y as the enzyme responsible for DNA replication in mitochondria [7,8].In the present work we have extended this enzymological study to the neuronal DNA polymerase /?.Here we show that 8-polymerase performs repairtype DNA synthesis induced by ultraviolet irradiation of neuronal nuclei. We further demonstrate that the properties of the neuronal enzyme are virtually indistinguishable from those previously established for all other mammalian /?-polymerases [12 -331. From

SUMMARYThe DNAs of eight parapoxviruses (four stomatitis papulosa viruses isolated from infected calves, a pseudocowpox virus isolated from a teat lesion of an infected cow and three orf viruses, one isolated from an infected sheep and two isolated from human infections) were analysed in CsC1 gradients. The mole of G+ C was calculated from the buoyant density and found to be approx. 63 % for all virus isolates examined. Parapoxvirus DNA thus has by far the highest G + C content of all poxvirus DNAs so far examined.Parapoxviruses form a group of morphologically dearly distinct viruses within the family Poxviridae. In contrast to the DNA of vaccinia virus (an orthopoxvirus) which is well characterized by its high mol. wt. of 122 X 10 6, (Geshelin & Berns, I974), presence of crosslinks at the termini (Berns & Silvermann, I97O; Geshelin & Berns, 1974) and a low G+ C content of 36 to ' 37 ~ (Joklik, I962a, b), very little is known about the genome of parapoxviruses. We have recently shown that the DNA of stomatitis papulosa virus has a tool. wt. of approx. 85 × Io 6 and that the ends are also cross-linked (Menna et ak I979).In order to characterize further the DNA of parapoxviruses we have calculated the mole of G + C of the DNAs of several isolates of parapoxviruses from the buoyant density obtained in analytical CsCl gradients.The following eight parapoxviruses were used in tfiis study: four stomatitis papulosa viruses (from infected calves), three orf viruses (one isolate from an infected sheep and two isolates from human infections) and one pseudocowpox virus (isolated from a teat lesion of an infected cow). The buoyant densities of the DNAs of the parapoxviruses was compared to the DNAs from two members of the orthopoxvirus genus, vaccinia (strain Elstree) and rabbitpox virus (strain Utrecht). The two orthopoxviruses were grown on the chorioallantoic membrane of developing chick embryos and the virions purified as described by Joklik 0962a). The parapoxvirus isolates were all grown on bovine foetal lung cells (Goldsmit & Barzilai, 1975). After virus adsorption for I h at 37 °C the cultures were further incubated until 90 ~ of the cells showed a c.p.e. The cultures were then frozen and thawed and the cells and viruses pelleted by ultracentrifugation. Virions were purified essentially as described by Joklik (1962a) for orthopoxviruses. Since the parapoxviruses banded in the upper third of the sucrose gradients in preliminary experiments, the procedure was slightly modified. Virions were pelleted through a cushion of 36 ~o sucrose for 2o rain at 26000 rev/min in a Beckman SW 40 rotor and banded in 25 to 4o ~ sucrose gradients for 25 rain at 23000 rev/min, also in a Beckman SW 40 rotor. In order to isolate the DNA of orthopoxviruses and parapoxviruses, virions were collected from sucrose gradients, pelleted by centrifugation for 3o min at 2500o g and resuspended in 50 mM-tris-HC1, pH 7"8, I mM-EDTA, 27 ~ sucrose and o'5 ~ SDS. The DNA was isolated by treatment of the virus suspension with 2o #g of proteinase K per ...