Cdc7-Dbf4 and the human S checkpoint response to UVC.

The S checkpoint response to ultraviolet radiation (UVC) that inhibits replicon initiation is dependent on the ATR and Chk1 kinases. Downstream effectors of this response, however, are not well characterized. Data reported here eliminated Cdc25A degradation and inhibition of Cdk2-cyclin E as intrinsic components of the UVC-induced pathway of inhibition of replicon initiation in human cells. A sublethal dose of UVC (1 J/m(2)), which selectively inhibits replicon initiation by 50%, failed to reduce the amount of Cdc25A protein or decrease Cdk2-cyclin E kinase activity. Cdc25A degradation was observed after irradiation with cytotoxic fluences of UVC, suggesting that severe inhibition of DNA chain elongation and activation of the replication checkpoint might be responsible for the UVC-induced degradation of Cdc25A. Another proposed effector of the S checkpoint is the Cdc7-Dbf4 complex. Dbf4 interacted weakly with Chk1 in vivo but was recognized as a substrate for Chk1-dependent phosphorylation in vitro. FLAG-Dbf4 formed complexes with endogenous Cdc7, and this interaction was stable in UVC-irradiated HeLa cells. Overexpression of FLAG- or Myc-tagged Dbf4 abrogated the S checkpoint response to UVC but not ionizing radiation. These findings implicate a Dbf4-dependent kinase as a possible target of the ATR- and Chk1-dependent S checkpoint response to UVC.

Effects of UV dose on formation of spontaneously developing pocks in Streptomyces azureus ATCC14921.

Spontaneously developing pocks (S pocks) of Streptomyces azureus ATCC14921 were formed by the both functions of conjugative plasmid pSA1 and lysogenic phage SAt2. The formation was affected by the dose of UV irradiation. The mean pock diameter in cultures treated with UV light at 0, 7.1, 14.2 and 21.3 x 10(2) microW. erg/cm, respectively, were 1.3, 0.4, 2.2, and 0.5 mm. The dose affected conjugative plasmid pSA1 related to pock formation. There was UV damage of autonomous pSA1 replicon and UV induction of the chromosomal integrated sequence. Increases and decreases in the amount of autonomous pSA1 replicon corresponded to increases and decreases, respectively, in the diameter of the pocks. Both pSA1 and SAt2 syntheses were developed in the large pocks (1.3 and 2.2 mm), but only SAt2 synthesis was developed in the pinhole pocks (0.4 and 0.5 mm).

Ionizing irradiation effects on S-phase in checkpoint mutants of the yeast Saccharomyces cerevisiae.

In mammalian cells, gamma-irradiation activates checkpoint controls to delay entry into, or passage through S-phase, while chronic exposure to methyl methanesulfonate or hydroxyurea causes a similar delay in yeast. In yeast, at least five genes are involved: RAD9, RAD17, RAD24, RAD53 and MEC1, a homologue of ATM. Here, using flow cytometry analysis and alkaline sucrose gradient centrifugation of labeled, newly made DNA, we demonstrate, in synchronized RAD wild-type Saccharomyces cerevisiae cells, that: (1) gamma-irradiation at START delays entry into S-phase, (2) irradiation shortly before or during early S-phase delays completion of S-phase and (3) the latter response is largely a consequence of replicon initiation inhibition. The delay produced by irradiation during early S-phase depends on the function of the checkpoint genes RAD9, RAD17, RAD24, RAD53, MEC1 and MEC3. However, at least four, RAD17, RAD53, MEC1, MEC3, are not needed to delay S-phase progression when cells are irradiated shortly before S-phase begins.

[Reorganization of DNA replicating system induced by 5-fluorodeoxyuridine and caffeine as the basis for radiosensitivity in human calls]. / Reorganizatsiia DNK-replitsiruiushchei sistemy, indutsiruemaia 5-flordesoksiuridinom i kofeinom, kak osnova radiochuvsvitel'nosti kletok cheloveka.

Treatment of diploid human fibroblasts with 5-fluorodeoryuridine (FUdR) and caffeine results in the increase in cellular radiosensitivity in terms of survival and chromosomal aberrations, on the one hand, and in radioresistant DNA synthesis (RDS), on the other hand, i.e. rather mimics those in mutant cells from patients with AT, XPII, Down syndrome, and others. A study was made of the autoradiographic length of simultaneously active adjacent replicon clusters. After incubation of diploid human cells with FUdR (10(-6) M, 6 h), this parameter was shown to reduce by two-fold, remaining unchanged upon 5 Gy irradiation. In contrast, after incubation of the cells with caffeine (2 mM, 30 min), this parameter was longer, compared to that in intact cells; upon 5 Gy irradiation the values remained almost the same as in the control. A possible relation of the data to the cellular radiosensitivity and RDS in the cells incubated with FUdR and caffeine is discussed.

Radiation down-regulates replication origin activity throughout the S phase in mammalian cells.

An asynchronous culture of mammalian cells responds acutely to ionizing radiation by inhibiting the overall rate of DNA replication by approximately 50% for a period of several hours, presumably to allow time to repair DNA damage. At low and moderate doses, this S phase damage-sensing (SDS) pathway appears to function primarily at the level of individual origins of replication, with only a modest inhibition of chain elongation per se. We have shown previously that the majority of the inhibition observed in an asynchronous culture can be accounted for by late G1cells that were within 2-3 h of entering the S period at the time of irradiation and which then fail to do so. A much smaller effect was observed on the overall rate of replication in cells that had already entered the S phase. This raised the question whether origins of replication that are activated within S phase per se are inhibited in response to ionizing radiation. Here we have used a two-dimensional gel replicon mapping strategy to show that cells with an intact SDS pathway completely down-regulate initiation in both early- and late-firing rDNA origins in human cells. We also show that initiation in mid- or late-firing rDNA origins is not inhibited in cells from patients with ataxia telangiectasia, confirming the suggestion that these individuals lack the SDS pathway.

Decreased number of simultaneously operating adjacent clusters of replicons in some human strains with and without X-irradiation.

Several parameters of DNA replicons and replicon clusters have been examined using DNA fiber autoradiography in normal vs. mutant human cell lines showing increased chromosomal sensitivity to ionizing radiation as well as radioresistant DNA synthesis. Rates of synthesis of individual replicons for unirradiated ataxia telangiectasia (AT) AT5MO, basal cell naevus syndrome (BCNS) BCN1SP and Down's Syndrome LCH944 appeared to be in the range seen with two normal fibroblast lines. With the longer labelling times (60-165 min), the average track lengths were longer in normal fibroblasts than mutant cell; after 5 Gy of radiation, normal and mutant cells had similar track lengths for all labelling times (10-165 min), as well as unchanged rates of replicon synthesis. These observations led to the determination of 'chain length', which measures simultaneously active adjacent replicon clusters. The main finding is that 'chain length' in mutant lines was significantly lower than that in the normal fibroblasts; upon 5 Gy irradiation, the values in normal cells were reduced about two-fold while the values for mutant cells remained about the same as controls. Thus, the experiments suggest that in unirradiated mutant cells DNA replication is delayed in a comparable manner as that induced by ionizing radiation in normal cells. A possible relation of the data to the chromosomal radiosensitivity and radioresistant DNA synthesis in the mutant lines is discussed.

Decreased rates of replicon initiation in mammalian cells.

We have designed an assay to measure the rate of initiation of DNA synthesis in Friend erythroleukemia cells and have shown that this parameter is reduced by gamma-radiation and treatment with 4'-demethyl-epipodophyllotoxin-9-(4,6-O-ethylene-beta-D-glucopyranoside) (VP-16). It is concluded, that double-strand breaks in DNA are the immediate cause for this effect. The decrease in the rate of replicon initiation is affected differently by different agents such as cis-diamminedichloroplatinum(II), cycloheximide, staurosporine, and 3-aminobenzamide. The analysis of these results indicates that the observed partial decrease of the rate of DNA initiation is most probably transmitted from the site of damage to the initiation site by one or more phosphorylation/dephosphorylation steps. It does not require de novo synthesis of protein factors, but is probably dependent on poly(ADP-ribosyl)ation of chromatin at the site of DNA breaks.

Radiation effects on DNA synthesis in a defined chromosomal replicon.

It has recently been shown that the tumor suppressor p53 mediates a signal transduction pathway that responds to DNA damage by arresting cells in the late G1 period of the cell cycle. However, the operation of this pathway alone cannot explain the 50% reduction in the rate of DNA synthesis that occurs within 30 min of irradiation of an asynchronous cell population. We are using the amplified dihydrofolate reductase (DHFR) domain in the methotrexate-resistant CHO cell line, CHOC 400, as a model replicon in which to study this acute radiation effect. We first show that the CHOC 400 cell line retains the classical acute-phase response but does not display the late G1 arrest that characterizes the p53-mediated checkpoint. Using a two-dimensional gel replicon-mapping method, we then show that when asynchronous cultures are irradiated with 900 cGy, initiation in the DHFR locus is completely inhibited within 30 min and does not resume for 3 to 4 h. Since initiation in this locus occurs throughout the first 2 h of the S period, this result implies the existence of a p53-independent S-phase damage-sensing pathway that functions at the level of individual origins. Results obtained with the replication inhibitor mimosine define a position near the G1/S boundary beyond which cells are unable to prevent initiation at early-firing origins in response to irradiation. This is the first direct demonstration at a defined chromosomal origin that radiation quantitatively down-regulates initiation.

[The relation of the characteristics of DNA replication to cell sensitivity to ionizing radiation in xeroderma pigmentosum in form II (XP2CP)]. / Sviaz' osobennostei replikatsii DNK s chuvstvitel'nost'iu kletok k ioniziruiushchei radiatsii pri pigmentnoi kseroderme v forme II (XP2CP).

Using DNA fiber autoradiography, DNA replication in cultured fibroblasts, derived from normal donors, and from XPII patient with increased sensitivity to ionizing radiation was estimated. Here evidence is provided on the fact that the fork movement, significantly decreased in XPII cells before irradiation, remains the same after exposure to X-rays. The density of replicon clusters simultaneously operating in tandem groups, which is initially much less in XPII cells compared to normal cells, also remains unchanged after exposure to X-rays (5 Gy), since the inhibition of DNA replication occurs to individual replicons only. Our data suggest that the inhibition of DNA replication in normal cells throughout the whole cluster, that drastically reduces the rate of DNA-chain growth, may provide an additional time to restore damaged chromosomes, i. e. it is part of the cellular defence mechanism. It seems likely that XPII cells are deficient in such a defence mechanism.

Effect of UV light on sister-chromatid exchanges, activation of alternative sites of replicon initiation and thymidine incorporation in CHO AA8, UV61 and UV5 cells.

The relative importance of the UV-induced pyrimidine(5-6)pyrimidine and the pyrimidine(6-4)-pyrimidone lesions in sister-chromatid exchanges (SCEs), activation of alternative sites of replicon initiation and thymidine incorporation were examined using wild-type Chinese hamster ovary (CHO) AA8 cells which remove both lesions, mutant CHO UV61 cells which remove only the (6-4) lesion and mutant CHO UV5 cells which remove neither lesion. Our data suggest that both lesions play a role in each end point examined. The relative importance of these lesions is dependent on the end point studied as well as the fluence used. For SCE induction and the activation of alternative sites of replicon initiation, the (6-4) lesion appears to play a predominant role, while for the thymidine incorporation studies the (6-4) lesion appears to play the predominant role at low fluences while the role of the (5-6) lesion increases at higher fluences.

Effect of UV light on DNA chain growth and replicon initiation in xeroderma pigmentosum variant cells.

The effects of UV light on DNA replication were examined in wild-type and xeroderma pigmentosum (XP) variant cells using DNA fiber autoradiography. Replication segments were significantly shorter in UV-exposed XP variant cells than they were in UV-exposed wild-type human cells immediately after exposure. This is consistent with data obtained by others using alkaline sucrose gradients suggesting that there is more blockage of DNA fork progression at UV-induced lesions in XP variant cells. With time, (2.5-5.0 h) the lengths of replication segments increased in both cell lines, suggesting that some type of bypass was occurring in XP variant cells or that excision repair was removing the blocking lesions. To determine if the post-replication defect in XP variant cells involved a failure to activate alternative sites of replicon initiation, high/low specific activity labeling was performed. The results obtained indicated that XP variant cells were able to activate alternative sites of replicon initiation. Therefore the unique phenotype of the XP variant cells is probably due to some other defect.

[The action of ionizing radiation on DNA replication in the cells of a healthy human subject and of a patient with Down's syndrome]. / Deistvie ioniziruiushchei radiatsii na replikatsiiu DNK v kletkakh zdorovogo cheloveka i bol'nogo s sindromom Dauna.

Using DNA fiber autoradiography we have revealed a new defect earlier unknown in Down's syndrome but analogous to that earlier shown by the authors in AT and basal cell nevus. This syndrome involves a significantly decreased number of simultaneously operating groups of replicons compared to that in normal cells., the rate of fork movement and the fusion of neighbouring units in the group being unchanged. Ionizing radiation (5 Gy) does not change the rate of DNA chain growth in the cells derived from the affected individuals, however, it significantly reduces this parameter in normal cells due to inhibition of replicon initiation in the whole clusters. Thus, radioresistant DNA synthesis in chromosomal instability syndromes may be explained by some defect in DNA replication in unirradiated cells analogous to that in irradiated normal cells.

Effects of UV light on DNA chain growth and replicon initiation in human cells.

Exposure of mammalian cells to 254 nm UV light produces lesions that block DNA polymerases at least on the leading strand. For rodent cells the extent and duration of this blockage is both cell line- and fluence-dependent. Using DNA fiber autoradiography we report here similar findings for human cells. Wild-type human cells did not exhibit significant blockage following exposure to 2.5 J/m2. After exposure to 5.0 J/m2, there was significant blockage immediately after exposure, but by 5 h segment lengths returned to control values. Excision-deficient human cells, on the other hand, exhibited significant blockage both immediately and 5.0 h after exposure to 2.5 J/m2. Exposure of rodent cells to UV light is also known to activate alternative sites of replication. Such activation would enable cells to replicate areas of DNA which do not contain a 'normal' site of initiation, yet contain blocking lesions both upstream and downstream. We have previously shown (Griffiths and Ling, 1987) that this activation is more pronounced and long-lived in excision-deficient Chinese hamster ovary (CHO) cells than it is in wild-type CHO cells. We report here that excision-deficient human cells also exhibited a marked and prolonged activation of alternative sites of replicon initiation. Wild-type human cells, on the other hand, exhibited little if any activation.

UV irradiation inhibits initiation of DNA replication from oriC in Escherichia coli.

Irradiation of Escherichia coli with UV light causes a transient inhibition of DNA replication. This effect is generally thought to be accounted for by blockage of the elongation of DNA replication by UV-induced lesions in the DNA (a cis effect). However, by introducing an unirradiated E. coli origin (oriC)-dependent replicon into UV-irradiated cells, we have been able to show that the environment of a UV-irradiated cell inhibits initiation of replication from oriC on a dimer-free replicon. We therefore conclude that UV-irradiation of E. coli leads to a trans-acting inhibition of initiation of replication. The inhibition is transient and does not appear to be an SOS function.

The scrapie disease process is unaffected by ionising radiation.

The incubation period of scrapie, its degenerative neuropathology and the replication of its causal unconventional virus are all tightly controlled parameters of the experimental disease in mice. Each parameter can vary depending on the strain and dose of virus, on the route of infection, and on the host genotype. Exposure to whole-body gamma-irradiation from Caesium 137 has no effect on the progress or development of the disease, based on the three independent indices of incubation period, neuropathology, or infectibility by high or low doses of virus. These results are based on an extensive series of experiments in many mouse strains and are consistent using different strains (ME7, 22A, 79A, 87V) and doses of virus, routes of infection, timing and dose of radiation (3-15 Gy) administered as single or fractionated exposures with or without bone-marrow (b.m.) replacement therapy. Levels of infection in the spleen are unaltered after lethal whole-body irradiation of the scrapie-infected host, despite several-fold reductions in tissue mass due to the loss of proliferating myeloid and lymphoid precursor cells and their progeny. Contrary to our earlier suggestion, scrapie infection with the 22A virus does not reduce the effectiveness of post-exposure bone-marrow replacements to recolonise an infected host after repeated ionising radiation totalling 15Gy. This work narrows the search for the candidate cells and biosynthetic systems which replicate the virus in the lymphoreticular and central nervous systems. Many programmed cellular events are radiation sensitive but protein synthesis is extremely radioresistant.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of alternative sites of replicon initiation in Chinese hamster cells exposed to ultraviolet light.

Exposure to UV light is known to produce lesions that block DNA polymerases at least on the leading strand. If several lesions are present in adjacent replicons, it is likely that sections of DNA would remain unreplicated because of the presence for blocking lesions. For cells to multiply and survive these areas must eventually be replicated. One mechanism that has been postulated to be involved in the replication of DNA between two blocking lesions is the activation of alternative sites of replicon initiation. To detect the existence of alternative sites of replicon initiation we employed the high specific/low specific activity labeling protocol first used by Huberman and Riggs (1968) for DNA fiber autoradiography. After development of the autoradiographs, the distances between adjacent sites of replicon initiation (inter-origin distances) were measured. In both wild-type Chinese hamster ovary (CHO) cells and UV-5 CHO cells, which exhibit no excision repair abilities, the inter-origin distances were, on average, shorter in cells exposed to UV, indicating that exposure to UV results in the activation of alternative sites of initiation. This activation appears to occur immediately after UV in both cell lines, but persist for a longer time in the excision-deficient line.

DNA synthesis in irradiated mammalian cells.

One of the first responses observed in S phase mammalian cells that have suffered DNA damage is the inhibition of initiation of DNA replicons. In cells exposed to ionizing radiation, a single-strand break appears to be the stimulus for this effect, whereby the initiation of many adjacent replicons (a replicon cluster) is blocked by a single-strand break in any one of them. In cells exposed to ultraviolet light (u.v.), replicon initiation is blocked at fluences that induce about one pyrimidine dimer per replicon. The inhibition of replicon initiation by u.v. in Chinese hamster cells that are incapable of excising pyrimidine dimers from their DNA is virtually the same as in cells that are proficient in dimer excision. Therefore, a single-strand break formed during excision repair of pyrimidine dimers is not the stimulus for inhibition of replicon initiation in u.v.-irradiated cells. Considering this fact, as well as the comparative insensitivity of human ataxia telangiectasia cells to u.v.-induced inhibition of replicon initiation, we propose that a relatively rare lesion is the stimulus for u.v.-induced inhibition of replicon initiation.

Inhibition of mammalian cell DNA synthesis by ionizing radiation.

A semi-log plot of the inhibitory effect of ionizing radiation on the rate of DNA synthesis in normal mammalian cells yields a two-component curve. The steep component, at low doses, has a D0 of about 5 Gy and is the result of blocks to initiation of DNA replicons. The shallow component, at high doses, has a D0 of greater than or equal to 100 Gy and is the result of blocks to DNA chain elongation. The target size for the inhibition of DNA replicon initiation is about 1000 kb, and the target size for inhibition of DNA chain elongation is about 50 kb. There is evidence that the target for both components is DNA alone. Therefore, the target size for inhibition of DNA chain elongation is consistent with the idea that an effective radiation-induced lesion in front of the DNA growing point somehow blocks its advance. The target size for inhibition of DNA replicon initiation is so large that it must include many replicons, which is consistent with the concept that a single lesion anywhere within a large group (cluster) of replicons is sufficient to block the initiation of replication of all replicons within that cluster. Studies with radiosensitive human cell mutants suggest that there is an intermediary factor whose normal function is necessary for radiation-induced lesions to cause the inhibition of replicon initiation in clusters and to block chain elongation; this factor is not related to poly(ADP-ribose) synthesis. Studies with radiosensitive Chinese hamster cell mutants suggest that double-strand breaks and their repair are important in regulating the duration of radiation-induced inhibition of replicon initiation but have little to do with effects on chain elongation. There is no simple correlation between inhibition of DNA synthesis and cell killing by ionizing radiation.

Altered DNA synthesis in irradiated and unirradiated ataxia-telangiectasia cells.

DNA synthesis in cells from ataxia-telangiectasia (AT) patients differs from that in normal cells in two principal ways. In unirradiated cultures, AT cells have a lower inherent rate of DNA synthesis (and, therefore, a longer S phase) than normal cells and, in cultures exposed to ionizing radiation, DNA synthesis is not inhibited as it is in normal cells; this radioresistant DNA synthesis is due to completely resistant DNA chain elongation and partially resistant DNA replicon initiation. It is probable that the defects in DNA synthesis in irradiated and unirradiated cells are related and are at least partially involved in the pathogenesis of this syndrome.

Subchromosomal DNA synthesis in synchronous V-79 chinese hamster cells after X irradiation.

A substantial fraction of replicon initiation events in Chinese hamster V-79 cells have been shown to be refractory to the effects of X irradiation immediately after exposure. This study examines the possibility that the initiation radiorefractive portion is the result of changes in replicon radiosensitivity as a function of position in S phase. The data obtained from DNA fiber autoradiograms and kinetic incorporation of radiolabeled thymidine from cells irradiated at various positions in S phase showed only slight changes in the proportion of replicons refractive to X irradiation immediately after exposure. These results indicate that initiation radiorefractive replicons may be an intrinsic property of V-79 cells and that cell-cycle-specific heterogeneity in radiation response cannot fully account for this phenomenon. The results also indicate that delayed inhibition of initiation events may play a larger role in the observed radiorefractive fraction than previously thought.