DNA strand breaks in resistant and sensitive murine lymphoma cells detected by the hydroxyapatite chromatographic technique.

Strand breaks were determined in L5178YS and L5178YR cell lines with DOS of 0.75 and 1.75 Gy, respectively, for 7 MeV electrons. The hydroxyapatite chromatographic technique was used to measure the breaks produced by 7 MeV electrons or 7 MeV neutrons immediately after irradiation or after maximal repair. Oxygen enhancement values for survival as well as strand breaks were determined for both cell lines and both qualities of radiation. The results indicate that despite a differential response to the lethal effects of radiation the levels of DNA strand breaks induced in these two lines were identical. Furthermore the values obtained for oxygen enhancement ratios (o.e.r.) and relative biological effectiveness (r.b.e.) for DNA strand breaks were different from those for cell survival. These results show that the difference in radiation sensitivity for cell killing is not reflected by the extent of DNA strand breaks measured by this method.     

Recovery of DNA synthesis after ultraviolet irradiation of xeroderma pigmentosum cells depends on excision repair and is blocked by caffeine

Normal human and xeroderma pigmentosum (XP, excision-defective group A) cells (both SV40-transformed) pulse-labeled with [(3)H]thymidine at various times after irradiation with ultraviolet light showed a decline and recovery of both the molecular weights of newly synthesized DNA and the rates of synthesis per cell. At the same ultraviolet dose, both molecular weights and rates of synthesis were inhibited more in XP than in normal cells. This indicates that excision repair plays a role in minimizing the inhibition of chain growth, possibly by excision of dimers ahead of the growing point. The ability to synthesize normal-sized DNA recovered more rapidly than rates of synthesis in normal cells, but both parameters recovered in phase in XP cells. During recovery in normal cells there are therefore fewer actively replicating clusters of replicons because the single-strand breaks involved in the excision of dimers inhibit replicon initiation. XP cells have few excision repair events and therefore fewer breaks to interfere with initiation, but chain growth is blocked by unexcised dimers. In both cell types recovery of the ability to synthesize normal-sized DNA was prevented by growing cells in caffeine after irradiation, possibly because of competition between the DNA binding properties of caffeine and replication proteins.Our observations imply that excision repair and semiconservative replication interact strongly in irradiated cells to produce a complex spectrum of changes in DNA replication which may be confused with parts of alternative systems such as post-replication repair.     

Hybridization of Chinese hamster cells sensitive to ultraviolet irradiation

Resistance to UV-light was studied in two UV-sensitive aneuploid Chinese hamster cell clones to different origin and degree of sensitivity, their respective polyploids and somatic cell hybrids. The karyotype of the parental clones, cell hybrids and polyploids was analyzed in parallel. A great variability of karyotypes was detected in hybrid cells. Serial cultivation of hybrids was accompanied by chromosome loss. Soon after fusion the hybrid clones proved to be more resistant to UV than the parental sensitive cells. However, their sensitivity increased with passages. The comparison of UV-sensitivity with data on karyotype analysis allowed to assume that the increase in sensitivity was correlated with the loss of particular chromosomes or chromosome regions. The results obtained indicated the existence of a polygenic control of UV-sensitivity, the multiple genes being assigned to different chromosomes. A reverse effect of ploidy was detected, i.e. a decrease in the resistance to the lethal action of UV-light in polyploids as compared to the parental clones.     

Correlation among the rates of dimer excision, DNA repair replication, and recovery of human cells from potentially lethal damage induced by ultraviolet radiation.

The kinetics of excision repair in confluent cultures of diploid human fibroblasts after ultraviolet irradiation at varying doses was measured by three different methods: (a) removal of thymine-containing dimers, (b) DNA excision repair synthesis, and (c) biological recovery of cells from the potentially lethal effects of the irradiation. Each method gave similar results and indicated that the excision rate was dependent upon the number of thymine-containing dimers induced (substrate concentration). For example, at a dose of 40 J/m2 (0.2% dimerization), the repair rate was 1.6 J/m2 per h as determined by a modified method to measure the number of thymine-containing dimers remaining in DNA and 1.65 J/m2 as measured by excision repair synthesis. At a dose of 7.5 J/m2, the repair rate was 0.5 J/m2 per h as measured by biological recovery, and at a dose of 7 J/m2, the repair rate was 0.46 J/m2 per h as measured by excision repair synthesis.     

Deoxyribonucleic acid excision repair in chromatin after ultraviolet irradiation of human fibroblasts in culture.

We have exposed confluent normal human fibroblasts to ultraviolet (UV) fluences of 5, 14, or 40 J/m2 and monitored the specific activity of post-UV repair synthesis in chromatin with [3H]thymidine pulses. We have shown that under conditions where no semiconservative deoxyribonucleic acid (DNA) synthesis is detectable, the specific activity of repair label in micrococcal nuclease resistant (core particle) DNA is about one-fifth that in bulk DNA at all three UV fluences. On the other hand, the distribution of thymine-containing pyrimidine dimers in bulk and nuclease-resistant regions measured either immediately after irradiation or at later times showed no significant differences; preferential labeling of linker (nuclease-sensitive) DNA during repair synthesis is thus apparently not due to a predominance of UV-induced photoproducts in linker relative to core particle DNA in the nucleosome. Pulse and pulse--chase experiments at 14 or 40 J/m2 with normal human or repair-deficient xeroderma pigmentosum (XP) cells showed that at most 30% of repair label in all these cells shifts from nuclease-sensitive (linker) DNA to nuclease-resistant (core particle) DNA.     

The duration of recovery and DNA repair in excision deficient derivatives of Escherichia coli K-12 after ultraviolet irradiation

Summary Our results indicate that cells of excision deficient (uvr) mutants of Escherichia coli K-12 which survive exposure to ultraviolet radiation may require several hours to complete their recovery. For example, the duration of the recovery period for cells exposed to 63 ergs mm^-2 at 254 nm was about 5 hours, the equivalent of slightly more than 4 generations of the unirradiated controls. During the recovery period the rate of cell division was reduced (Figs. 3 and 4), the cells gradually regained resistance to complex medium (Figs. 1 and 3), and they became refractory to photoreactivation (Fig. 1). Over the same period of time their pattern of DNA synthesis changed. More intact molecules, similar to those found in unirradiated controls, and relatively fewer discontinuous molecules were synthesized (Figs. 6 and 7).     

Genomic integrity of T1 DNA after gamma-and ultraviolet irradiation.

T1 DNA, gamma-irradiated in the phage particle or irradiated with ultraviolet light was checked for structural integrity by kinetics of melting and reannealing. gamma-Irradiated DNA differed in all thermokinetic properties by a factor of 3-4 from DNA degraded by mechanical or enzymatical treatments. Ultraviolet irradiation caused much smaller effects than gamma-irradiation. Considering the frequency of pyrimidine dimers in relation to the gamma-ray induced lesions, strong evidence can be derived, that in addition to single base damages, local denatured regions are produced by gamma-irradiation. Such regions, formed possibly by direct absorption of radiation energy in DNA, i.e. by primary ionizations, are associated with base lesions and are passed over during reannealing.     

Replicative bypass repair of ultraviolet damage to DNA of mammalian cells: Caffeine sensitive and caffeine resistant mechanisms

Replicative bypass repair of UV damage to DNA was studied in wide variety of human, mouse and hamster cells in culture. Survival curve analysis revealed that in established cell lines (mouse L, Chinese hamster V79, HeLa S3 and SV40-transformed xeroderma pigmentosum (XP)), post-UV caffeine treatment potentiated cell killing by reducing the extrapolation number and mean lethal UV fluence (Do). In the Do reduction as the result of random inactivation by caffeine of sensitive repair there were marked clonal differences among such cell lines, V79 being most sensitive to caffeine potentiation. However, other diploid cell lines (normal human, excision-defective XP and Syrian hamster) exhibited no obvious reduction in Do by caffeine. In parallel, alkaline sucrose sedimentation results showed that the conversion of initially smaller segments of DNA synthetized after irradiation with 10 J/m² to high-molecular-weight DNA was inhibited by caffeine in transformed XP cells, but not in the diploid human cell lines. Exceptionall, diploid XP variants had a retarded ability of bypass repair which was drastically prevented by caffeine, so that caffeine enhanced the lethal effect of UV. Neutral CsCl study on the bypass repair mechanism by use of bromodeoxyuridine for DNA synthesis on damaged template suggests that the pyrimidine dimer acts as a block to replication and subsequently it is circumvented presumably by a new process involving replicative bypassing following strand displacement, rather than by gap-filling de novo. This mechanism worked similarly in normal and XP cells, whether or not caffeine was present, indicating that excision of dimer is not always necessary. However, replicative became defective in XP variant and transformed XP cells when caffeine was present. It appears, therefore, that the replicative bypass repair process is either caffeine resistant or sensitive, depending on the cell type used, but not necessarily on the excision repair capability.     

DNA repair patch-mediated double strand DNA break formation in human cells

To investigate the mechanism of double strand DNA break formation in mammalian cells, an in vitro assay was established using closed circular DNA containing two uracils on opposite DNA strands (18 and 30 base pairs apart) and extracts prepared from human cells. In this assay, formation of double strand breaks was detected by the conversion of circular DNA to linear DNA. Approximately 4-fold more double strand DNA breaks were produced by extracts from cells deficient in DNA ligase I (46BR) relative to those produced by extracts from control cells (MRC5, derived from a clinically normal individual). In parallel with the amount of double strand DNA breaks, extracts from 46BR cells produced longer repair patches (up to 24 bases in length) than those from MRC5 cells (typically <5 bases long). When purified DNA ligase I was added to 46BR extracts to complement the DNA ligase deficiency, only a negligible difference was found between the amount of doublestrand DNA breaks or the repair patch size generated in the assay relative to MRC5 extracts. Together, our data demonstrate that double strand DNA breaks are produced through formation of DNA repair patches. We refer to this process of double strand break formation as the "DNA repair patch-mediated pathway."     

Theory of co-operative formation of defects in DNA molecule under ultraviolet irradiation

The process of thymine dimers formation in DNA under the action of ultraviolet irradiation has been analyzed theoretically taking into account the long range transfer of electronic excitation along the molecule. First, for the case of homopolymer (poly dA.dT) an exact analytical solution of the problem has been obtained. For the case of random walks of excitation as well as for the case of its directional motion the distribution functions of undamaged regions on their lengths have been calculated. These functions turned out to differ drastically from a trivial exponential distribution taking place without excitation's transfer. At the same time the transfer of excitation leads to the effect of thymine dimers clustering—the new dimers are formed mainly in the vicinity of dimers formed earlier. The degree of clustering γ (the mean number of dimers per cluster) depends quadratically on the concentration of clusters C in the homopolymer for the case of random walks of excitation. For the case of a heteropolymer having inhomogeneous distribution of potential dimers (two thymines situated one by one in the same strand) a machine algorithm for numerical solution of the problem by the Monte-Carlo method has been proposed. Calculations for the heteropolymer with a random sequence of bases have shown that in this case the curve γ(c) is S-shaped and its form agrees with the experimental one obtained by Shafranovskaya, Trifonov, Lazurkin &; Frank-Kamenetskii (1972). The values of parameters providing the best agreement between theory and experiment have been obtained. Using these values of parameters the calculations of distribution function of undamaged regions and curves γ(c) for different concentrations of AT-pairs in DNA have been performed. These calculations show that the degree of clustering γ rises dramatically with increasing concentration of AT-pairs in DNA. Theoretical predictions which can be tested experimentally have been summarized.