Elimination of stress-induced activation of DNA reparative synthesis in the myocardium by increasing the load on the heart

The rate of DNA reparative synthesis was studied in the nucleus of myocardial cells in the heart compensatory hyperfunction (HCH) induced by the aorta coarctation and in animals exposed to surgical stress without the aorta coarctation. It was established that both surgery and emotional painful stress activated the DNA reparative synthesis in myocardial cells. For example, 12-24 hrs following the surgery the DNA reparation rate exceeded control values by 50-74%. HCH induced no changes in the DNA reparation rate in nuclei of myocardial cells. The mechanism of stress-induced DNA damage is discussed and a hypothesis is put forward on stabilizing the effect of hyperfunction on the DNA structure.     

Requirements for adenosine triphosphate in DNA repair in isolated hepatic nuclei

ATP appeared to play several roles during DNA repair in isolated hepatic nuclei. It stimulated carcinogen-activated reparative DNA synthesis, it prevented degradation of newly synthesized DNA and it suppressed the accumulation of single-strand breaks in DNA. The results suggest DNA repair is an energy-dependent process using DNA polymerase, DNA-dependent ATPase and DNA ligase.     

Weakening of the ultraviolet ray-induced unscheduled DNA synthesis in human lymphocytes in extreme old age

The intensity of unscheduled DNA synthesis was studied in UV-irradiated (10--15 J/m2) peripheral blood lymphocytes of 80--90 years old persons. In these extreme old age persons, reparative DNA synthesis was found sufficiently reduced in comparison with that in middle aged (20--43 years old) ones. The role of DNA repair processes in ageing is under discussion.     

Effect of tryptophan on isolated hepatocytes of rats

The addition of tryptophan to adult rat hepatocyte cultures stimulated DNA synthesis. The increase in DNA synthesis as measured by 3H-thymidine incorporation into DNA was observed on treatment of the cultures with tryptophan for 48 h but also as short as for 6 h in comparison with control cultures. An increase was also apparent at 30 h which was maintained for up to 48 h post treatment with tryptophan. The increase in DNA synthesis by tryptophan cannot be attributed to cell injury or to increased DNA degradation. Of the degradative enzymes added after harvesting the hepatocytes, only DNase decreased incorporation of 3H-thymidine. The observed effect was specific for tryptophan since treatment with kynurenine, isoleucine, methionine or serine failed to show a significant effect. Pretreatment of cultured hepatocytes with hydroxyurea prevented the tryptophan stimulated increase in DNA synthesis suggesting that the latter was due to replicative and not to reparative DNA synthesis. Experiments performed with the addition of diethylnitrosamine also alluded to tryptophan's role in replicative DNA synthesis. The mechanism of tryptophan-induced DNA synthesis is discussed.     

Possible role of SV40 T antigen in cell transformation]

The effect of purified SV40 T antigen on DNA synthesis in isolated nuclei from the confluent culture of CV-1 cells was studied. In the presence of T antigen the incorporation of [3H]TTP into DNA was found to be 2 to 3 times as high as in the control nuclei. The resulting labelled DNA was subjected to alkaline sucrose gradient centrifugation, which revealed the presence of 4S DNA species, corresponding to Okazaki fragments of animal cells. The latter finding suggests a replicative mode of DNA synthesis induced by T antigen. T antigen isolated from the cells infected with SV40 tsA-mutant and kept at a nonpermissive (41 degrees) temperature fails to stimulate DNA synthesis in isolated nuclei from resting cells. On storage at 4 degrees SV40 T antigen gradually loses its ability to stimulate DNA synthesis and by the 8th day even suppresses it when tested on isolated nuclei from a growing cell culture. No effect of T antigen on the endonuclease-induced reparative synthesis of DNA could be observed. The data described suggest that T antigen is directly involved in the control of DNA synthesis in the cells infected or transformed with SV40.     

Unscheduled DNA synthesis in isolated hepatic nuclei after treatment of rats with methylnitrosourea in vivo

Administration of the carcinogenic methylating agent, methylnitrosourea, to rats caused a significant increase in endogenous DNA synthesis assayed subsequently in isolated hepatic nuclei $\text{in}\text{vitro}$. DNA synthesis was related directly to the dose of carcinogen and inversely to the interval between treatment and isolation of nuclei. This synthesis appears to represent the continuation $\text{in}\text{vitro}$ of unscheduled, reparative DNA synthesis initiated in damaged cells $\text{in}\text{vivo}$.     

Increased sensitivity for detection of carcinogen-induced DNA repair with the chain terminator dideoxythymidine.

The sensitivity for quantitation of DNA excision repair caused by a carcinogen is increased when the resynthesis of DNA is inhibited with dideoxythymidine (ddThd), a chain terminator. Incorporation of ddThd in primary liver cell cultures leaves discontinuities in the DNA chain at sites of reparative synthesis in response to benzo(a)pyrene [B(a)P] treatment. This is detectable by the accumulation of cellular DNA with reduced molecular weight on alkaline sucrose gradients. Up to 78 percent of the DNA in cells incubated with B(a)P and ddThd had greatly reduced molecular weight compared to control cells, whereas only 32 percent of the DNA in cells treated with B(a)P but not incubated with ddThd was affected similarly.     

Autoradiographic Study of Gamma-Ray Induced Unscheduled DNA Synthesis in Bean Root Meristem Cells

The gamma-ray induced unscheduled DNA synthesis in root meristem cells of Vica faba was studied autoradiographically by calculating the number of cells with different 3H-thymidine labelling degree. It was found that the level of unscheduled synthesis in cells with intermediate dose (500 R) irradiation was higher than that in cells with lower dose (250 R) irradiation; however, higher dose (1000 R) irradiation would inhibit the reparative replication.     

Dynamic component of maintaining genomic stability in murine bone marrow cells after chronic low-intensity irradiation lasting one year

For analysis of a dynamic component state of the system of maintenance of genome stability, which represent a condition of its expression (first of all, genes of the control of phases of cell cycle, the DNA repair and redox systems) after a long chronic exposure to a small dose, the activity of replicative, reparative DNA synthesis, DNA damage as well as oxyradical content in the bone marrow cells of mice (critical for radiation effects mammalian system) after 1 year radiation exposure to a dose 63.7 cGy (0.17 cGy/day) were studied. The considerable enhancement of replicative and reparative DNA synthesis activity by 67% (p = 0.0033) and 60% (p = 0.000004) accordingly in relation to the control and some, but statistically not significant (p = 0.149) tendency to increase (by 30%) the content of a superoxide anion-radical were established. Strong and highly significant correlation (r = 0.8681; P = 0.99975) between DNA damage and O2-. content in bone marrow cells of the irradiated mices, which indicate the large DNA damage by oxiradicals, probably, due to the loss of a part of structural proteins and conformation changes in expression sites of a chromatin, were detected. The obtained results interpreted as representing the change of a dynamic component state of a system of maintenance of genome stability, the epigenic transfer of that to descendants of the irradiated cells can be the cause for formation and maintenance of radiation-induced genome instability.     

Autoradiographic study of the rate of DNA synthesis in the duct epithelial cells of the epididymis and of the brain subependymal zone in the rat following whole-body x-ray irradiation

The experimental data have been analyzed on the labeled cell distribution related to the grain count over the nucleus in autographs of histological sections (5 mcm) made of the rat brain subependymal zone and of epididymis duct epithelium at different time after 3H-thymidine injection and X-irradiation in the dose of 300 cGr. These results served some additional grounds to the recently established conclusion that a repeated successive decrease in the rate of DNA synthesis is occurring in the cell system starting from stem cell (level I) to semistem cell (levels II-VII) (Gracheva, 1982 r). 5 days after irradiation, at the peak of reparative proliferation, the cell reproduction was intensified, these cells having normally both middle and high levels of DNA synthesis. This process is running of the background of the inhibition of reproduction of cells with the inherent low level of DNA synthesis which are to start differentiation after mitosis. All this makes for the increase in the mean grain count over the nuclei, without changes of the inherent rates of DNA synthesis in the successive generations of the stem cells.     

Stimulation with quaterin of DNA replication and repair

Quaterine [3-(2,2,3-trimethylhydrasinium)propionate] possessing a wide spectrum of physiological activity has been studied for its effect on the intensity of replicative and reparative DNA synthesis in different rat tissues (liver, thymus, heart, intestine mucosa and spleen) in order to investigate molecular mechanisms of its action. It is shown that the pronounced stimulation of DNA synthesis in all tissues, as a rule, takes place 1-6h after quaterine administration in doses of 25 and 100 mg/kg. The estimation of the given compound effect on DNA synthesis after its multiple administration to animals (for 5, 10, 15 days in a dose of 100 mg/kg) permits supposing that 3-(2,2,2-trimethylhydrasinium)propionate is able of providing either stable proliferation of cells (thymus, spleen) or their hyperplasia and polyploidization (heart, liver). The data obtained make it possible to explain (to some extent) quaterine ability to activate immune responses, to stimulate healing of wounds and burns.     

Effect of UV radiation on the DNA-membrane complex of Bacillus subtilis]

The influence of UV-light on DNA-membrane complex (DMC) of Bacillus subtilis was studied. An increased DNA content in DMC for strains 168 and rec A-, and a degradation of DMC for strain polA- have been registrated. The increase in DNA in DMC of the two former strains is inhibited by caffeine to be correlated with changes in protein content in DMC, determined by a radioactive label, but not with lipid content. Thus, the association of DNA with the membrane is mediated by proteins. DNA increasing capacity seen in DMC after UV-irradiation and after the following incubation of bacteria in the complete medium is correlated with a relative sensitivity of strains. To explain these data, it is supposed that the reparative synthesis is accomplished in cell on their membranes and that for the normal completion of DNA repair the association between DNA and the membrane is necessary.     

Activity profiles of enzymes that control the uracil incorporation into DNA during neuronal development.

We have shown that DNA polymerase beta, the only nuclear DNA polymerase present in adult neurons, cannot discriminate between dTTP and dUTP, having the same Km for both substrates. This fact suggests that during reparative DNA synthesis, in adult neurons, dUMP residues can be incorporated into DNA. Since uracil DNA-glycosylase functions to prevent the mutagenic effects of uracil in DNA coming as a product of deamination of cytosine residues or as a result of dUMP incorporation by DNA polymerase, we have studied the perinatal activity of uracil DNA-glycosylase and of 2 enzymes (nucleoside diphosphokinase and dUTPase) involved in dUTP metabolism. Our data indicate that during neuronal development there is a rapid decrease in uracil DNA-glycosylase which could impair the removal of uracil present in DNA in adult neurons. However, misincorporation of dUMP into DNA might be kept to a low frequency by the action of dUTPase present at all developmental stages.     

DNA repair endonuclease activity during synchronous growth of diploid human fibroblasts.

DNA-repair endonuclease activity in response to UV-induced DNA damage was quantified in diploid human fibroblasts after synchronizing cell cultures to selected stages of the cell cycle. Incubation of irradiated cells with aphidicolin, an inhibitor of DNA polymerases alpha and delta, delayed the sealing of repair patches and allowed estimation of rates of strand incision by the repair endonuclease. The apparent Vmax for endonucleolytic incision and Km for substrate utilization were determined by Lineweaver-Burk and Eadie-Hofstee analyses. For cells passing through G1, S or G2, Vmax for reparative incision was, respectively, 7.6, 8.4 and 8.4 breaks/10(10) Da per min, suggesting that there was little variation in incision activity during these cell-cycle phases. The Km values of 2.4-3.1 J/m2 for these cells indicate that the nucleotidyl DNA excision-repair pathway operates with maximal effectiveness after low fluences of UV that are in the shoulder region of survival curves. Fibroblasts in mitosis demonstrated a severe attenuation of reparative incision. Rates of incision were 11% of those seen in G2 cells. Disruption of nuclear structure during mitosis may reduce the effective concentration of endonuclease in the vicinity of damaged chromatin. The extreme condensation of chromatin during mitosis also may restrict the accessibility of reparative endonuclease to sites of DNA damage. Confluence-arrested fibroblasts in G0 expressed endonuclease activity with Vmax of 5.5 breaks/10(10) Da per min and a Km of 5.5 J/m2. The greater condensation of chromatin in quiescent cells may restrict the accessibility of endonuclease to dimers and so explain the elevated Km. When fibroblasts were synchronized by serum-deprivation, little variation in reparative endonuclease activity was discerned as released cells transited from early G1 through late G1 and early S. Proliferating fibroblasts in G1 were shown to express comparatively high numbers of reparative incision events in the absence of aphidicolin which was normally used to inhibit DNA polymerases and hold repair patches open. It was calculated that in G0, S and G2 phase cells, single-strand breaks at sites of repair remained open for 30, 19 and 14 sec, respectively. In G1 phase cells, repair sites remained open for 126 sec. Addition of deoxyribonucleosides to G1 cells reduced this time to 42 sec suggesting that the slower rate of synthesis and ligation of repair patches in G1 was due to a relative deficiency of deoxyribonucleotidyl precursors for DNA polymerase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Participation of the intracellular enzymes in the control of the mutation process

Summary The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive ³H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10–11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication — hydroxyurea — had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.     

Disordered DNA repair in tumor cells as affected by sarcolysine

Sarcolysine-induced damaging and reparative processes in the primary structure of tumour cell DNA have been studied. The presence of low sarcolysine concentrations (1 mkM) in the cell culture during the first two hours of incubation caused suturing of DNA molecules. The increase of the incubation time from 4 to 18 hours and the rise in the drug concentration (by 10.20 times and more) resulted in intensive accumulation of one-strand breaks. However, we have not observed the appearance of high-molecular DNA, which is the evidence of the completion of the reparative process. The impulse treatment with sarcolysine (1 hour, 10 mkM) with subsequent drug removal caused the irreversible damage of DNA reparative processes at the stage of short fragments' suturing.     

Genetic analysis of the Hsm3 protein domain structure in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Gene HSM3 encodes the Hsm3 protein involved in the minor branch in the system responsible for the correction of mismatched bases in DNA structure and controls replicative and reparative spontaneous mutagenesis in yeast Saccharomyces cerevisiae. Spontaneous and UV-induced mutagenesis was studied in three mutant alleles of gene HSM3, and repair effectivity of artificial heteroduplexes was assessed in DNA molecule. The resuts of these studies allowed establishment of the protein domain structure of protein Hsm3 and functions of each domain: the N-terminal domain is responsible for binding to mispaired bases, and the C-terminal domain ensures the interaction with other proteins involved in the system of mismatched base correction.     

Development of spontaneous mutations in somatic mamalian cells and DNA replication. II. Expression of gene mutations in cultured cells]

The dependence of spontaneous rate and phenotypic expression of 6-mercaptopurine resistance mutations of DNA replication synthesis was studied in cultured Chinese hamster cells. Spontaneous mutations arising with a constant rate per cell per time unit independently on DNA replication rate were shown to be expressed only in the course of subsequent cell divisions. The frequency of N-nitrosomethylurea induced mutations in cells with reduced and normal DNA replication rate is approximately the same. However, DNA replication synthesis is necessary for the phenotypic expression of both induced and spontaneous mutations. The causes of the phenotypic lag are discussed.     

The effect of UV irradiation and of UV-irradiated autologous blood on the functional state of human peripheral blood lymphocytes

The effect of UV irradiation (UVI, 254 nm) and of UV-irradiated autologous blood on the spontaneous and mitogen-induced DNA-synthetic activity of intact lymphocytes has been studied. Lymphocytes were isolated from nonirradiated and irradiated blood, and from the mixture of UV-irradiated blood with the intact one in the volume ratio close to that in the blood stream during UV-irradiated blood autotransfusion (1:10, 1:40, 1:160). It has been shown that UVI of the whole blood caused in some donors the increase in spontaneous DNA synthesis, while in others the decrease or no statistically significant changes were observed. The analysis of the results obtained shows an inverse relation of the UVI effect to the initial level of spontaneous DNA synthesis (r = -0.68). In contrast to direct UVI effect, an addition of UV-irradiated blood to the autologous intact one resulted in an increase in spontaneous DNA synthesis in lymphocytes of all the samples examined. A 7-day cocultivation of lymphocytes, isolated from irradiated and nonirradiated blood samples, revealed a 1.8 times increase compared to the calculated value. The mitogen-induced DNA synthesis has a low sensitivity to UV rays, since the mitogens and the irradiation of optical range have presumably the common targets. It is assumed that photomodification of HLA-D/DR antigens can be a trigger mechanism for activation of immunocompetent cells by UVI.