The effect of SSB-protein from Ehrlich ascites carcinoma cells on activity of various enzymes of DNA replication and repair

The effect of a single-stranded DNA-binding protein (SSB-protein) form Ehrlich ascites tumour cells (EAT) on the activity of homologous purified DNA-polymerases alpha and beta, DNA-replicase, primase and DNA-polymerases from phage T4 and Bacillus stearothermophillus was studied. It was shown that the SSB-protein caused a 1.5-2.5-fold stimulation of the DNA-polymerase alpha activity on different templates (e.g., denaturated and activated DNA, poly(dA). The degree of stimulation depended on the template type, protein/template ratio and purity of DNA-polymerase alpha. The activity of DNA-polymerase was inhibited by the SSB-protein, when the activated DNA was used as a matrix and was unchanged on the denaturated DNA. The activity of some prokaryotic DNA-polymerases was increased under the influence of the SSB-protein. The protein enhanced the processivity of T4 DNA-polymerase and strongly inhibited the activity of replicase and primase. A conclusion about the complex effect of the SSB-protein on the activity of replicative and repair enzymes is drawn.     

Mechanisms of the radiation-induced disorder of DNA synthesis. The correlation of biosynthesis, DNA repair and DNA polymerase alpha and beta activity in the bone marrow of rats exposed to gamma quanta and fast neutrons

A study was made of DNA biosynthesis and repair and alpha- and beta-DNA-polymerase activity in rat bone marrow during the first 24 hours following whole-body irradiation with gamma-quanta and fast neutrons (up to 6 Gy). There was a correlation between the post-irradiation inhibition of DNA biosynthesis, a decrease in DNA-polymerase activity and template reparability. The data obtained permitted to consider the radiation-induced disturbance of DNA biosynthesis and the change in beta-polymerase activity as one of the possible mechanisms of formation of high relative biological effectiveness of neutrons.     

The role of N-nitrosourea-induced changes in the nucleoid structure and activity of repair enzymes in the development of drug resistance in mice with leukemia L1210

Using centrifugation of the nucleoid in a neutral sucrose gradient, the damages in the secondary structure of DNA and the activity of repair enzymes, such as DNA-polymerases alpha and beta and poly(ADP-riboso) polymerase, induced by 1-methyl-nitrosourea (MNU) and 1.3-bis (2-chloroethyl)-1-nitrosourea (BCNU) injected at maximal nonlethal single doses to mice bearing parent leukemia cells (L1210/0) and resistant to MNU and BCNU leukemia L1210 cells (L1210/MNU and L1210/BCNU), were studied. The MNU-induced production of single-strand breaks in L1210/0 and L1210/MNU cells was more conspicuous in newly replicated DNA than in those in preexisting DNA. A more fast repair of the damages in newly replicated DNA was detected in L1210/BCNU and especially in L1210/MNU leukemia cells as compared with L1210/0 cells. The data obtained suggest that there are prone errors in the repair of DNA template, since most of the single-strand breaks were revealed in the newly replicated DNA synthesized on the repaired DNA. The repair of DNA damages in L1210/BCNU and especially in L1210/MNU cells was accompanied by the activation of DNA-polymerases alpha and beta and poly(ADP-riboso)polymerase. Both DNA-polymerases--alpha and beta--were shown to be involved in repair of DNA damages induced by MNU and only DNA-polymerase beta was involved in the repair of damages induced by BCNU.     

The role of DNA polymerases alpha, beta and gamma in nuclear DNA synthesis.

The effects of the inhibitors 2'3' dideoxythymidine triphosphate (ddTTP) and 1-beta-D-arabinofuranosyl cytosine triphosphate (araCTP) on DNA synthesis in isolated S-phase HeLa S3 nuclei have been examined. These effects are compared with the effects of the same inhibitors in partially purified preparations of DNA polymerases alpha and beta. The effect of ddTTP on partially purified DNA polymerase gamma was also tested. DNA polymerases beta and gamma were very sensitive to ddTTP whereas DNA polymerase alpha and DNA synthesis in isolated nuclei were quite resistant. The synthesis and subsequent ligation of primary DNA pieces ('Okazaki fragments') were not affected by the presence of this inhibitor. DNA synthesis in isolated nuclei and DNA polymerase alpha activity were very sensitive to araCTP whereas DNA polymerase beta was almost totally resistant to the inhibitor. The results indicate a major role for DNA polymerase alpha in DNA replication.     

Effect of exogenous DNA on the content and biosynthesis of endogenous DNA in rat bone marrow]

The influence of exogenous DNA on the content of endogenous DNA and the rate of biosynthesis was studied in rat bone marrow. After the injection of highly-polymerized homologous DNA to intact rats the content of rat DNA per 1 gm of the bone marrow decreased within the first 3 days (the most marked fall occurred in 3 days--by 36%), and returned to the normal by the 6th day. The rate of DNA biosynthesis in rat bone marrow increased in 18 hours (doublled in comparison with control), remained elevated within 6 days (by 58%) and approached the normal level from 1 to 3 days after the DNA injection.     

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: evidence that DNA polymerases delta and beta are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

The involvement of DNA polymerases alpha, beta, and delta in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase alpha) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors on MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [3H]thymidine incorporated into repaired DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 micrograms of aphidicolin/mL, 6% by 10 microM BuPdGTP, 13% by anti-(DNA polymerase alpha) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 micrograms of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase alpha) antibodies into HF nuclei. These results indicate that both DNA polymerases delta and beta are involved in repairing DNA damage caused by MNNG.     

Immunocytochemical localization of chick DNA polymerases alpha and beta +

An immunofluorescent method using specific antibodies was employed to detect DNA polymerases alpha and beta in chick cells. With monoclonal antibodies produced by four independent hybridoma clones, most of the DNA polymerase alpha was shown to be present in nuclei of cultured chick embryonic cells. With a polyclonal, but highly specific, antibody against DNA polymerase beta, this enzyme was also shown to be present in nuclei. DNA polymerase alpha was detected in proliferating cells before cell contact and in lesser amount in resting cells after cell contact, indicating that its content is closely correlated with cell proliferation. On the other hand, similar amounts of DNA polymerase beta were detected in proliferating and resting cells. Furthermore, DNA polymerase beta was detected in nuclei of most cells, while DNA polymerase alpha was detected only in large round nuclei in seminiferous tubules of chick testis. DNA polymerase alpha is presumably present in cells that are capable of DNA replication, and during the cell cycle it seems to remain in the nuclei during the G1, S, and G2 phases, but to leave from condensed chromatin for the cytoplasm during the mitotic phase.     

Further characterization of a cell-free system for measuring replicative and repair DNA synthesis with cultured human fibroblasts and evidence for the involvement of DNA polymerase alpha in DNA repair.

DNA repair synthesis can be specifically measured in osmotically opened, confluent cultured human fibroblasts after exposure to DNA damaging agents such that both induction and mediation of DNA repair synthesis can take place in this cell-free system. Alternatively, by utilizing osmotically shocked, log phase cells and altering the DNA precursors, pH and ionic strength, replicative DNA synthesis can be specifically monitored. Autoradiographic studies show that virtually all of the nuclei from the lysates of the confluent, UV-iradiated cells are lightly labeled in the fashion characteristic of DNA repair. By contrast, only a fraction of nuclei is labeled in a population of unperturbed, opened log phase cells and the labeling is heavy and characteristic of replicative synthesis. Furthermore, equilibrium density gradient sedimentation shows that DNA synthesis in lysates of log-phase cells is semiconservative, whereas that with UV-irradiated cells is repair synthesis. This open cell system has been used to study the enzymology of DNA repair. Thus, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerases beta and gamma, does not inhibit either replicative or repair synthesis. By contrast, aphidicolin, a specific inhibitor of DNA polymerase alpha, inhibits DNA repair and replicative synthesis in both intact and permeabilized cells. Finally, phage T4 UV-exonuclease stimulates repair synthesis, but only when phage T4 UV-endonuclease is also added to the UV-irradiated nuclei.     

The roles of DNA polymerases alpha, beta, and gamma in DNA repair synthesis induced in hamster and human cells by different DNA damaging agents

The involvement of DNA polymerases alpha, beta, and gamma in DNA repair synthesis was investigated in subcellular preparations of cultured hamster and human cells. A variety of DNA damaging agents, including bleomycin, neocarzinostatin, UV irradiation, and alkylating agents, were utilized to induce DNA repair. The sensitivity of repair synthesis, as well as replicative synthesis and purified DNA polymerase beta activity, to inhibition by the DNA polymerase inhibitors dideoxythymidine triphosphate, aphidicolin, cytosine arabinoside triphosphate, and N-ethylmaleimide was determined. No evidence was obtained for a major role of polymerase gamma in any type of repair synthesis. In both hamster and human cells, the sensitivity of bleomycin- and neocarzinostatin-induced repair synthesis to ddTTP inhibition was essentially identical with that observed for purified polymerase beta, indicating these repair processes proceeded through a mechanism utilizing polymerase beta. Repair synthesis induced by UV irradiation and alkylating agents was not sensitive to ddTTP, indicating repair of these lesions occurred through a pathway primarily utilizing a different DNA polymerase; presumably polymerase alpha. However, replicative synthesis was much more sensitive to polymerase alpha inhibitors than was repair synthesis induced by UV irradiation or alkylating agents. Neither the amount of DNA damage nor the amount of induced repair synthesis influenced the degree to which the different DNA polymerases were involved in repair synthesis. The possibility that "patch size" or the actual type of DNA damage determines the extent to which different polymerases participate in DNA repair synthesis is discussed.     

Evidence that DNA polymerases alpha and beta participate differentially in DNA repair synthesis induced by different agents

The roles of DNA polymerases alpha and beta in DNA replication and repair synthesis were studied in permeable animal cells, using different agents to induce repair synthesis. DNA polymerase inhibitors were used to investigate which polymerases were involved in repair synthesis and in replication. Polymerase alpha was responsible for replication. On the other hand, both polymerases alpha and beta were involved in DNA repair synthesis; the extent to which each polymerase participated depended primarily on the agent used to damage DNA. Polymerase beta was primarily responsible for repair synthesis induced by bleomycin or neocarzinostatin, whereas polymerase alpha played a more prominent role in repair synthesis indiced by N-methyl-N'-nitro-N-nitrosoguanidine or N-nitrosomethyl urea. More DNA damage was induced by the alkylating agents than by bleomycin or neocarzinostatin, suggesting that the extent of involvement of polymerase alpha or beta in DNA repair synthesis is related to the amount or type of DNA damage. In addition, salt concentration was found to have little or no effect on the results obtained with the DNA polymerase inhibitors. Our findings provide an explanation for conflicting reports in the literature concerning the roles of DNA polymerases alpha and beta in DNA repair.     

2',3'-Dideoxythymidine 5'-triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis in human cells: evidence for involvement of DNA polymerase delta

It is well established that DNA replication and ultraviolet-induced DNA repair synthesis in mammalian cells are aphidicolin-sensitive and thus are mediated by one or both of the aphidicolin-sensitive DNA polymerases, alpha and/or delta. Recently, it has been shown that DNA polymerase delta is much more sensitive to inhibition by the nucleotide analogue 2',3'-dideoxythymidine 5'-triphosphate (ddTTP) than DNA polymerase alpha but is less sensitive than DNA polymerase beta [Wahl, A. F., Crute, J. J., Sabatino, R. D., Bodner, J. B., Marraccino, R. L., Harwell, L. W., Lord, E. M., & Bambara, R. A. (1986) Biochemistry 25, 7821-7827]. We find that DNA replication and ultraviolet-induced DNA repair synthesis in permeable human fibroblasts are also more sensitive to inhibition by ddTTP than polymerase alpha and less sensitive than polymerase beta. The Ki for ddTTP of replication is about 40 microM and that of repair synthesis is about 25 microM. These are both much less than the Ki of polymerase alpha (which is greater than 200 microM) but greater than the Ki of polymerase beta (which is less than 2 microM). These data suggest that DNA polymerase delta participates in DNA replication and ultraviolet-induced DNA repair synthesis in human cells.     

Involvement of deoxyribonucleic acid polymerase beta in nuclear deoxyribonucleic acid synthesis.

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.     

DNA-polymerase activity and DNA synthesis in the hepatocyte nuclear matrix

The comparative analysis of DNA-synthetase activity of hepatocytes, isolated nuclei and nuclear matrix from normal and regenerating rat liver was performed. The highest enrichment with newly-synthesized DNA was registered in the DNA fraction associated with the nuclear matrix both in vivo and in vitro. The functioning of DNA polymerases alpha and beta in the matrix was shown. Our results indicate that DNA polymerase beta is more firmly bound with the nuclear matrix in the cells of normal liver but this enzyme is eluted almost completely from the nuclei of regenerating liver cells. At the first moment after gamma-irradiation of rats the preferential initiation of unscheduled DNA synthesis in vivo has been observed on the nuclear skeletal structures. This may serve as an indication on the possibility that DNA repair process occurs on the nuclear matrix.     

Effects of aphidicolin and/or 2',3'-dideoxythymidine on DNA repair induced in HeLa cells by four types of DNA-damaging agents

The alkaline sucrose density gradient centrifugation method was modified to permit detection of 1 lesion/10(9) daltons of DNA. With this technique, the involvements of DNA polymerases in DNA repair of damage by dimethyl sulfate, UV irradiation, neocarzinostatin, and bleomycin were studied in HeLa cells with the aid of the DNA polymerase inhibitors aphidicolin and 2',3'-dideoxythymidine. DNA repair after UV-induced damage seemed to involve only polymerase alpha, while repair of damage by the other three agents involved both polymerase alpha and a non-alpha polymerase, probably polymerase beta. But repair after damage by dimethyl sulfate differed from that after damage by neocarzinostatin or bleomycin with respect to the co-operations of polymerase alpha and polymerase beta: in repair of dimethyl sulfate-induced damage, both polymerases operated on the same lesions, whereas after damage by neocarzinostatin or bleomycin, polymerase alpha and polymerase beta functioned independently on different lesions.     

Cytoplasmic localization of human repetitive DNA revealed by in situ hybridization

Previously, we showed that a human repetitive DNA sequence (Sau3A family) belonging to a satellite DNA is unstable and constantly excised from the chromosomes (R. Kiyama, H. Matsui, and M. Oishi, 1986, Proc. Natl. Acad. Sci. USA 83, 4665). The unusual property of the repetitive DNA, along with another repetitive DNA (Alu sequence), was further investigated by in situ hybridization in several different human cells including HeLa, bone marrow, and peripheral blood cells. We found that the excised repetitive DNA sequences are localized not only in nuclei, but also in cytoplasm. These results have confirmed the instability of these DNA sequences in the chromosomes and further suggest that the alpha satellite DNA and the Alu sequence which were excised from the chromosomes are released from nuclei to cytoplasm.     

DNA polymerase beta from brain neurons is a repair enzyme.

DNA polymerase beta was isolated from rat cortex neurons and characterised. Its properties were strikingly similar to those of other mammalian beta-polymerases. In adult rats, this was the major DNA polymerase occurring in neuronal nuclei, which contained no alpha-polymerase, 99.2% beta-polymerase and only 0.8% gamma-polymerase. Isolated neuronal nuclei of this developmental stage were shown to perform ultraviolet-induced repair DNA synthesis in vitro. Since beta-polymerase was virtually the exclusive DNA polymerase in these nuclei it was concluded that the beta 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 beta-polymerase inhibitor. However, the presence of minute amounts of gamma-polymerase in neuronal nuclei and its susceptibility to d2TTP did not allow one to rule out an ancillary role of DNA polymerase gamma in DNA repair. In view of the similarity of the neuronal DNA polymerase beta with all other mammalian beta-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 beta-polymerases.     

Differentiation of lens and neural cells in chicken embryos is accompanied by simultaneous decay of DNA replication machinery

DNA polymerase alpha was detected in cells of developing chicken embryos by an immunofluorescent method using a monoclonal antibody specific for the high molecular weight polypeptide of chicken DNA polymerase alpha, and DNA polymerase beta was detected using a rabbit anti-chicken DNA polymerase beta antibody. In lens tissue of the 3- to 4-day chicken embryo, fluorescence with anti-DNA polymerase alpha antibody was detected in nuclei of lens epithelial cells but not in nuclei of lens fiber cells which had differentiated from epithelial cells. The localization of cells containing DNA polymerase alpha coincided with the distribution of cells capable of DNA replication as detected by [3H]thymidine autoradiography. Similar results were obtained during the differentiation of neural matrix cells to neuroblasts in the developing neural tube. In contrast to DNA polymerase alpha, DNA polymerase beta was detected in nuclei of both undifferentiated and differentiated cells of these tissues. Since the disappearance of DNA polymerase alpha was very rapid after the onset of differentiation, the DNA replication machinery in which DNA polymerase alpha plays a central role is thought to decay almost simultaneously with the onset of cellular differentiation in these tissues.     

Spatial distribution of DNA loop attachment and replicational sites in the nuclear matrix

Biochemical fractionation was combined with high resolution electron microscopic autoradiography to study the localization in rat liver nuclear matrix of attached DNA fragments, in vivo replicated DNA, and in vitro synthesized DNA. In particular, we determined the distribution of these DNA components with the peripheral nuclear lamina versus more internally localized structural elements of isolated nuclear matrix. Autoradiography demonstrated that the bulk of in vivo newly replicated DNA associated with the nuclear matrix (71%) was found within internal matrix regions. A similar interior localization was observed in isolated nuclei and in situ in whole liver tissue. Likewise, isolated nuclear lamina contained only a small amount (12%) of the total matrix-bound, newly replicated DNA. The structural localization of matrix-bound DNA fragments was examined following long-term in vivo labeling of the DNA. The radioactive DNA fragments were found predominantly within interior regions of the matrix structure (77%), and isolated nuclear lamina contained less than 15% of the total nuclear matrix-associated DNA. Most of the endogenous DNA template sites for the replicative enzyme DNA polymerase alpha (approximately 70%) were also sequestered within interior regions of the matrix. In contrast, a majority of the endogenous DNA template sites for DNA polymerase beta (a presumptive repair enzyme) were closely associated with the peripheral nuclear lamina. A similar spatial distribution for both polymerase activities was measured in isolated nuclei before matrix fractionation. Furthermore, isolated nuclear lamina contained only a small proportion of total matrix-bound DNA polymerase alpha endogenous and exogenous template activities (3-12%), but a considerable amount of the corresponding beta polymerase activities (47-52%). Our results support the hypothesis that DNA loops are both anchored and replicated at nuclear matrix-bound sites that are predominantly but not exclusively associated with interior components of the matrix structure. Our results also suggest that the sites of nuclear DNA polymerase beta-driven DNA synthesis are uniquely sequestered within the characteristic peripheral heterochromatin shell and associated nuclear envelope structure, where they may potentially participate in DNA repair and/or replicative functions.     

Quinolone antibiotics inhibit eucaryotic DNA polymerase alpha and beta, terminal deoxynucleotidyl transferase but not DNA ligase

DNA polymerases alpha and beta, Terminal deoxynucleotidyl Transferase and DNA ligases from chicken thymus were purified to homogeneity. Quinolone antibiotics (nalidixic acid, oxolinic acid and pefloxacin ) known to inhibit DNA replication were tested for their effects on these enzymes. DNA ligase activity was not affected by the three drugs. DNA polymerases alpha and beta were inhibited by competitive mechanisms. Surprisingly, Terminal deoxynucleotidyl Transferase was strongly inhibited by the three compounds and more efficiently by nalidixic acid. The significance of these results is discussed in terms of the possible involvement of the enzymes in the respective DNA replication and repair processes.     

Studies on DNA polymerases of Xenopus laevis oocytes: subcellular distribution and physical association of DNA polymerase alpha inside the nucleus

The localization of DNA polymerases in Xenopus laevis oocytes and eggs was studied. Oocytes have a high level of DNA polymerase alpha activity detected exclusively in the nuclei, while mitochondria contain all the DNA polymerase activity of the gamma type. No DNA polymerase beta activity is present in the nuclear fraction. Moreover, the beta activity is not present in unfertilized eggs. In oocytes, DNA polymerase alpha is weakly bound to the nucleoplasm. The leakage of the enzyme from whole nuclei can be prevented using polyvinylpyrrolidone, a nuclear pore sealing agent.