Recovery of the Ability to Synthesize DNA in Segments of Normal Size at Long Times After Ultraviolet Irradiation of Human Cells

DNA synthesized in human cells within the first hour after ultraviolet (UV) irradiation is made in segments of lower molecular weight than in nonirradiated cells. The size of these segments approximates the average distance between pyrimidine dimers in the parental DNA. This suggests that the dimers interrupt normal DNA synthesis and result in gaps in the newly synthesized DNA. However, DNA synthesized in human cells at long times after irradiation is made in segments equal or nearly equal to those synthesized by nonirradiated cells. The recovery of the ability to synthesize DNA in segments of normal size occurs in normal human cells, where the dimers are excised, and also in cells of the human mutants xeroderma pigmentosum (XP), where the dimers remain in the DNA. This observation implies that the pyrimidine dimer may not be the lesion that causes DNA to be synthesized in smaller than normal segments.     

Repair,replication and survival in UV-irradiatedEscherichia coli

The influence of dimer removal through excision or photoreactivation on the kinetics of DNA synthesis, sedimentation profiles of DNA molecules and survival of cells was investigated in excision-deficient and excision-proficientEscherichia coli K-12 after a flux of 20 J m⁻². In excision-deficient cells photoreactivation did not influence the kinetics of DNA synthesis for a long period and the sedimentation properties of DNA synthesized immediately after photoreactivation were influenced only slightly. However, survival was increased remarkably. In excision-proficient cells where dimers were removed through excision, the kinetics of DNA synthesis increased rapidly, normal-sized DNA molecules were synthesized 60 min after irradiation and survival was substantially higher than in the above-mentioned case. This can hardly be interpreted as a more complete repair of dimers by excision because the persistence of dimers in these cells did not significantly influence either the kinetics of DNA synthesis or normalization of DNA molecules and/or survival of cells. It is concluded that persisting dimers play an important role in excision-deficient but not in excision-proficient cells, that a non-dimer damage to DNA causes inhibition of DNA synthesis after UV and that this damage ia of primary importance for excision-proficient cells which can easily cope with persisting dimers.     

Effect of Caffeine on Postreplication Repair in Human Cells

DNA synthesized shortly after ultraviolet (UV) irradiation of human cells is made in segments that are smaller than normal, but at long times after irradiation the segments made are normal in size. Upon incubation, both the shorter and the normal segments are elongated and joined by the insertion of exogenous nucleotides to form high molecular weight DNA as in nonirradiated cells. These processes occur in normal human cells, where UV-induced pyrimidine dimers are excised, as well as in xeroderma pigmentosum (XP) cells, where dimers are not excised. The effect of caffeine on these processes was determined for both normal human and XP cells. Caffeine, which binds to denatured regions of DNA, inhibited DNA chain elongation and joining in irradiated XP cells but not in irradiated normal human or nonirradiated cells. Caffeine also caused an alteration in the ability to recover synthesis of DNA of normal size at long times after irradiation in XP cells but not in normal cells.     

DNA Repair in Potorous tridactylus

The DNA synthesized shortly after ultraviolet (UV) irradiation of Potorous tridactylis (PtK) cells sediments more slowly in alkali than that made by nonirradiated cells. The size of the single-strand segments is approximately equal to the average distance between 1 or 2 cyclobutyl pyrimidine dimers in the parental DNA. These data support the notion that dimers are the photoproducts which interrupt normal DNA replication. Upon incubation of irradiated cells the small segments are enlarged to form high molecular weight DNA as in nonirradiated cells. DNA synthesized at long times (~ 24 h) after irradiation is made in segments approximately equal to those synthesized by nonirradiated cells, although only 10-15% of the dimers have been removed by excision repair. These data imply that dimers are not the lesions which initially interrupt normal DNA replication in irradiated cells. In an attempt to resolve these conflicting interpretations, PtK cells were exposed to photoreactivating light after irradiation and before pulse-labeling, since photoreactivation repair is specific for only one type of UV lesion. After 1 h of exposure ~ 35% of the pyrimidine dimers have been monomerized, and the reduction in the percentage of dimers correlates with an increased size for the DNA synthesized by irradiated cells. Therefore, we conclude that the dimers are the lesions which initially interrupt DNA replication in irradiated PtK cells. The monomerization of pyrimidine dimers correlates with a disappearance of repair endonuclease-sensitive sites, as measured in vivo immediately after 1 h of photoreactivation, indicating that some of the sites sensitive to the repair endonuclease (from Micrococcus luteus) are pyrimidine dimers. However, at 24 h after irradiation and 1 h of photoreactivation there are no endonuclease-sensitive sites, even though ~ 50% of the pyrimidine dimers remain in the DNA. These data indicate that not all pyrimidine dimers are accessible to the repair endonuclease. The observation that at long times after irradiation DNA is made in segments equal to those synthesized by nonirradiated cells although only a small percentage of the dimers have been removed suggests that an additional repair system alters dimers so that they no longer interrupt DNA replication.     

The role of pyrimidine dimers in postreplication repair in Neurospora

Summary Using the Micrococcus luteus dimer specific endonuclease assay of Wilkins (1973), and photoreactivation we have examined the induction and fate of ultraviolet induced pyrimidine dimers in the excision defective strain, uvs-2, of Neurospora crassa.Dimer induction was fluence dependent from 0 to 800 ergs/mm² UV. An interdimer distance of 19.6x10⁶ DNA molecular weight was found after a fluence of 220 ergs/mm². We confirm the earlier report that this mutant is completely excision defective (Worthy and Epler 1972). Photoreactivation (PR), which greatly enhanced survival (by 10 fold after 440 ergs/mm² UV), reduced significantly (40–44%) the number of UV-endonuclease sensitive sites found in irradiated DNA. This treatment also alleviated immediately some of the temporary blocks to high molecular weight DNA synthesis (elongation or ligation) seen in irradiated cells.We have also attempted to elucidate the mechanism of cellular postreplication repair used to overcome the UV inhibition to DNA synthesis. It was determined that during postreplication repair, Neurospora does not use recombination to bypass dimers and that single stranded DNA gaps opposite dimers do not appear to be present during the time when DNA being synthesized is made only in short pieces.     

Correlation between survival,ability to rejoin DNA and stability of DNA after preirradiation inhibition of protein synthesis in arec - mutant ofEscherichia coli K12

A 90 min inhibition of protein synthesis induced by starvation for amino acids (AA^-) or by treatment with chloramphenicol (CAP) prior to UV irradiation (2.5 J m^-2) increased the resistance of the strainEscherichia coli K12 SR19 to UV radiation more than ten-fold. Under these conditions, cultures in which protein synthesis was inhibited before the UV irradiation rejoin short regions of DNA synthesized after the irradiation to a normal-size molecule, whereas an exponentially growing culture does not rejoin DNA synthesized after UV irradiation to a molecule of a normal size. In the exponentially growing culture both the parental and the newly synthesized DNA are unstable after the irradiation. In cultures with inhibited protein synthesis only the parental DNA is somewhat unstable. InEscherichia coli K12 SR19 where protein synthesis was inhibited before the irradiation, a correlation between the survival of cells, the ability to rejoin short regions of DNA synthesized after UV irradiation and a higher stability of both parental and newly synthesized DNAs could be demonstrated.     

Inhibition of DNA replication by ultraviolet light.

DNA replication in ultraviolet-irradiated HeLa cells was studied by two different techniques: measurements of the kinetics of semiconservative DNA synthesis, and DNA fiber autoradiography. In examining the kinetics of semiconservative DNA synthesis, density label was used to avoid measuring the incorporation due to repair replication. The extent of inhibition varied with time. After doses of less than 10J/m2 the rate was initially depressed but later showed some recovery. After higher doses, a constant, low rate of synthesis was seen for at least the initial 6 h. An analysis of these data indicated that the inhibition of DNA synthesis could be explained by replication forks halting at pyrimidine dimers. DNA fiber autoradiography was used to further characterize replication after ultraviolet irradiation. The average length of labeled segments in irradiated cells increased in the time immediately after irradiation, and then leveled off. This is the predicted pattern if DNA synthesis in each replicon continued at its previous rate until a lesion is reached, and then halted. The frequency of lesions that block synthesis is approximately the same as the frequency of pyrimidine dimers.     

Dependence of DNA dark repair on protein synthesis in Escherichia coli.

Summary We investigated the influence of aminoacidless treatments applied prior and after UV irradiation on survival, dimer excision, postirradiation DNA degradation, DNA synthesis and sedimentation profiles of parental DNA ofE. coli B/r Hcr⁺ cells. In dependence on the treatment applied, the fluence 50 J/m² yielded distinctly different fractions of survivors within 0,03–85%. In all cases dimers were completely excised. The rate of DNA degradation was similar during a 30–40 min period after UV during which the bulk of dimers was excised. Degradation ceased, however, earlier in the prestarved cells than in exponentially growing ones; it was prolonged by aminoacidless postincubation. Sedimentation profiles of parental DNA did not differ during the whole period of dimer excision. In cells DNA synthesis was not restored for several hours after addition of amino acids. In cells addition of amino acids resulted in a fast resumption of DNA synthesis. We conclude that removal of dimers and repair of gaps were similar in all cases. We believe that aminoacidless treatments influence production and repair of damage to the sites of DNA replication. The treatment appears to prevent this damage when applied before UV irradiation, but interferes with its restoration when applied after UV irradiation. Consequently, the former treatment increases survival of cells while the latter produces an opposite effects.     

Chromosome and DNA damage and their repair in higher plants irradiated with short-wave ultraviolet light

In this review the authors present only their own results. They include the determination of the duration of the different stages of the cell cylce in UV-irradiated barley cells, the effect of different UV doses on the frequency of chromosome aberrations in barley, the increase in UV-induced chromosome aberration frequency induced in barley by caffeine and the effect of UV doses on the induction of pyrimidine dimers and sites sensitive to UV-endonuclease action (ESS) in barley cells and Nicotina tabacum protoplasts. In addition, the excision of pyrimidine dimers and ESS after irradiation with various doses of UV, unscheduled DNA synthesis in N. tabacum protoplasts and the correlation between the induction of pyrimidine dimers in DNA and the frequency of chromosome aberrations are reported. Data demonstrating that photoreactivation decrease the number of DNA lesions and chromosome aberrations induced by UV are also presented.     

DNA repair and survival in UV-irradiated chicken embryo fibroblasts

The role of the pyrimidine dimer in cell killing, DNA synthesis and repair has been studied by utilizing the light-requiring DNA-repair mechanism of photo- reactivation in UV-irradiated chicken-embryo fibroblasts. Survival, as measured by colony-forming ability at 41°C, is increased in cells left in the light. The initial inhibition of DNA synthesis by UV is much less in light-treated cells, and levels reach that of unirradiated controls much faster than when the cells are left in the dark. The number of endonuclease-sensitive sites (dimers)_measured by an assay with a crude extract from M. luteus, rapidly decreases as the cells are allowed to photoreactive. However, in the dark, significant amounts of repair also occur, but at a much lower rate and with a lag phase of several hours. Unscheduled DNA synthesis occurs to a similarly low extent in both dark- and light-treated cells, confirming the finding that some amount of excision repair occurs that is light-independent. When survival is examined as a function of the number of dimers present, the dimers, not the non-dimer products, appear to be responsible for cell killing. In this study, the removal of dimers in vivo by photoreactivation has made it possible to demonstrate directly that dimers are primarily responsible for the deleterious effects of UV on DNA synthesis and survival.     

Significance of dimers to the size of newly synthesized DNA in UV-irradiated Chinese hamster ovary cells.

DNA synthesized after UV irradiation is smaller than that in unirradiated cells even when pulse-labeling times are increased to compensate for the overall reduction in the rate of DNA replication. By isolating newly replicated DNA, incubating it with dimer-specific endonuclease from Micrococcus luteus, and analyzing it on alkaline sucrose gradients, we have been able to demonstrate that this DNA is synthesized in segments corresponding in size to the interdimer distance on the parental strand. In addition, the same DNA analyzed on neutral gradients shows no reduction in molecular weight as a result of UV irradiation and/or endonuclease digestion. Our data are thus inconsistent with the presence of "gaps" in newly synthesized DNA opposite the dimers on the parental strand. We suggest that if such gaps are produced as a result of delayed synthesis around dimers, they are filled before the growing point reaches the next dimer.     

Postreplication repair of DNA in chick cells: studies using photoreactivation.

During replication of DNA after ultraviolet irradiation, gaps are left in the newly-synthesized DNA strands in both bacterial and animal cells and these gaps are subsequently sealed by a process known as postreplication repair. In order to test whether it is the ultraviolet-induced pyrimidine dimers which are responsible for the production of these daughter-strand gaps in animal cells, we have used chick embryo fibroblasts. In these cells the pyrimidine dimers are photoreactivable, i.e. they can be split by an enzymatic process dependent on visible or near ultraviolet light. Our results indicate that chick cells possess a postreplication repair system similar to that in mammalian cells; gaps are produced in the newly-synthesized strands and then filled in. If the ultraviolet-irradiated cells are first photoreactivated to remove most of the dimers, the number of daughter-strand gaps produced is much less than without photoreactivation. This suggests that the dimers are indeed responsible for the formation of many of the gaps in the newly-synthesized DNA. Ultraviolet light also inhibits the overall rate of DNA synthesis. This inhibition is, however, only partly overcome by photoreactivation.     

Fully efficient chromosome dimer resolution in Escherichia coli cells lacking the integral membrane domain of FtsK

In bacteria, septum formation frequently initiates before the last steps of chromosome segregation. This is notably the case when chromosome dimers are formed by homologous recombination. Chromosome segregation then requires the activity of a double‐stranded DNA transporter anchored at the septum by an integral membrane domain, FtsK. It was proposed that the transmembrane segments of proteins of the FtsK family form pores across lipid bilayers for the transport of DNA. Here, we show that truncated Escherichia coli FtsK proteins lacking all of the FtsK transmembrane segments allow for the efficient resolution of chromosome dimers if they are connected to a septal targeting peptide through a sufficiently long linker. These results indicate that FtsK does not need to transport DNA through a pore formed by its integral membrane domain. We propose therefore that FtsK transports DNA before membrane fusion, at a time when there is still an opening in the constricted septum.     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.     

Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts

The inhibition of DNA replication in ultraviolet-irradiated human fibroblasts was characterized by quantitative analysis of radiation-induced alterations in the steady-state distribution of sizes of pulse-labeled, nascent DNA. Low, noncytotoxic fluences (<1 J/m², producing less than one pyrimidine dimer per replicon) rapidly produced an inhibition of DNA synthesis in half-replicon-size replication intermediates without noticeably affecting synthesis in multi-repliconsize intermediates. With time, the inhibition produced by low fluences spread progressively to include multi-replicon-size intermediates. The results indicate that ultraviolet radiation inhibits the initiation of DNA synthesis in replicons. Higher (>1 J/m², producing more than one dimer per replicon) cytotoxic fluences inhibited DNA synthesis in operating replicons presumably because the elongation of nascent strands was blocked where pyrimidine dimers were present in template strands. Xeroderma pigmentosum fibroblasts with deficiencies in DNA excision repair exhibited an inhibition of replicon initiation after low radiation fluences. indicating the effect was not solely dependent upon operation of the nucleotidyl excision repair pathway. Owing to their inability to remove pyrimidine dimers ahead of DNA growing points, the repair-deficient cells also were more sensitive than normal cells to the ultraviolet-induced inhibition of chain elongation. Xeroderma pigmentosum cells belonging to the variant class were even more sensitive to inhibition of chain elongation than the repair-deficient strains despite their ability to remove pyrimidine dimers. This analysis suggests that normal and repair-deficient human fibroblasts either are able to rapidly bypass certain dimers or these dimers are not recognized by the chain elongation machinery.     

DNA Chain Elongation and Joining in Normal Human and Xeroderma Pigmentosum Cells after Ultraviolet Irradiation

DNA synthesized in human cells after ultraviolet (UV) irradiation is made in segments of lower molecular weight than in unirradiated cells. Within several hours after irradiation these smaller units are both elongated and joined together. This repair process has been observed in normal human fibroblasts, HeLa cells, and fibroblasts derived from three types of xeroderma pigmentosum patients—uncomplicated with respect to neurological problems, complicated (de Sanctis-Cacchione syndrome), and one with the clinical symptoms of xeroderma pigmentosum but with normal repair replication. The ability of human cells to elongate and to join DNA strands despite the presence of pyrimidine dimers enables them to divide without excising the dimers present in their DNA. It may be this mechanism which enables xeroderma pigmentosum cells to tolerate small doses of UV radiation.     

DNA synthesis in pretreated and ultraviolet-irradiated cells ofEscherichia coli B/rHcr + andHcr −

DNA synthesis after the ultraviolet irradiation was followed in the excision proficient strainEscherichia coli B/rHcr ⁺, in which the ability to excise thymin dimers was suppressed by a preirradiation inhibition of DNA and protein syntheses and in the excision deficient strainEscherichia coli B/rHcr ^?. Synthesis of pulse-labeled DNA, its stability and semiconservative DNA synthesis were compared in both strains. It was found that cells of theHcr ⁺ strain restore semiconservative DNA synthesis and the pulselabeled DNA appears stable, in spite of the fact that dimers are not excised under these conditions. On the other hand, cells of theHcr ^? strain are unable to restore semiconservative DNA synthesis and the pulselabeled DNA is degraded. As the repair by the excision of dimers under the used experimental conditions may be excluded in both strains, it is possible to assume that activity of enzymes included in theHcr ⁺ marker is prerequisite for restoring the DNA synthesizing system in theHcr ⁺ strain.     

Differentiation of metaxylem cell line in the root ofAllium cepa L.

Summary The differentiation processes of the metaxylem cell line in the root ofAllium cepa are characterized by amplification phenomena of repetitive DNA sequences mainly localized in heterochromatic regions of metaphase chromosomes. Moreover, these sequences are heavily methylated. This paper presents additional results on variation in endogenous DNA methylation in different developing root segments. The results show that methylation is higher in apical meristematic cells than the differentiating segments; contrastingly, total RNA synthesis seems to be correlated with undermethylation. Addition of labelled methyl groups to DNA by eukaryotic methylase, DNA digestions with different restriction enzymes specific for methylated sites and HPLC analysis confirmed the above results. Moreover, variation in methylation levels during differentiation occur not only at the internal cytosine of the-CCG-sites, but also at external cytosine. Furthermore, methylation affects other sites containing the trinucleotides-CXG-. In conclusion, root differentiation inAllium cepa seems to be correlated with gene activation modulated by the methylation/demethylation of particular DNA sequences.     

Replicative intermediates in UV-irradiated simian virus 40

We have used Simian virus 40 (SV40) as a probe to study the replication of UV-damaged DNA in mammalian cells. Viral DNA replication in infected monkey kidney cells was synchronized by incubating a mutant of SV40 (tsA58) temperature-sensitive for the initiation of DNA synthesis at the restrictive temperature and then adding aphidicolin to temporarily inhibit DNA synthesis at the permissive temperature while permitting pre-replicative events to occur. After removal of the drug, the infected cells were irradiated at 100 J/m2 (254 nm) to produce 6-7 pyrimidine dimers per SV40 genome, and returned to the restrictive temperature to prevent reinitiation of replication from the SV40 origin. Replicative intermediates (RI) were labeled with [3H]thymidine, and isolated by centrifugation in CsCl/ethidium bromide gradients followed by BND-cellulose chromatography. The size distribution of daughter DNA strands in RI isolated shortly after irradiation was skewed towards lengths less than the interdimer spacing in parental DNA; this bias persisted for at least 1 h after irradiation, but disappeared within 3 h, by which time the size of the newly-synthesized DNA exceeded the interdimer distance. No significant excision of dimers from parental strands in either replicative intermediates or Form I (closed circular) DNA molecules was detected. These data are consistent with the hypothesis that replication forks are temporarily blocked by dimers encountered on the leading strand side of the fork, but that daughter strand continuity opposite dimers is eventually established. Evidence was obtained for the generation at late times after irradiation, of Form I molecules in which the daughter DNA strands contain dimers. Thus DNA strand exchange as well as trans-dimer synthesis may be involved in the generation of supercoiled Form I DNA from UV-damaged SV40 replicative intermediates.     

Photoreactivation and repair replication in rat kangaroo cells

Potorous tridactylis cells can perform photoreactivation, i.e., the visible light- catalyzed reversal of UV-induced pyrimidine dimers in DNA. UV-induced inhibitions of total RNA and DNA synthesis can also be partially reversed by exposure to visible light. P. tridactylis cells can also perform repair replication, but the extent of the latter is reduced if the cells are exposed to visible light (VL). None of these effects are observed in mouse L cells, which cannot perform photoreactivation. The results are consistent with the concept of pyrimidine dimers are one of the main substrates for repair replication.