Low-level DNA exchanges in normal human and xeroderma pigmentosum cells after UV irradiation

We have used a T4 endonuclease V assay method for UV-induced pryrimidine dimers in cellular DNA in vivo to obtain evidence for recombinational DNA exchanges after UV irradiation of normal human and Xeroderma pigmentosum (XP) cells. Our data indicate that the endonuclease-sensitive sites in excision-defective XP cells are removed very slowly from the irradiated parental strands and appear concomitantly in daughter strands newly synthesized during post-UV incubation. In the defective XP cells, the extent of appearance of sensitive sites in daughter strands synthesized during a period of 24 h after 10 J/m² appears to be small, probably less than 15% of the initial number of sensitive sites detected in cellular parental strands. Demonstration of such exchanges between normal-density parental and 5-bromodeoxyuridine-labeled daughter strands by alkaline CsCl isopycnic centrifugation was unsuccessful. Further, the extent is much lower in normal human cell because of their efficiet excision repair of the dimers before and after exchanges than in the defective XP cells.     

T4-endonuclease V-sensitive sites in DNA from ultraviolet-irradiated human cells.

Human diploid cells (WI38) were pre-labeled with 32Pi, exposed to ultraviolet irradiation and then pulse labeled with [3H]thymidine. The extracted DNA from these cells was subsequently treated with the T4-endonuclease V, an enzyme which specifically nicks DNA strands at positions adjacent to pyrimidine dimers. Sedimentation in alkaline sucrose gradients revealed that the DNA synthesized after irradiation, as well as that made before, contained endonuclease-sensitive sites. Our results suggest that pyrimidine dimers are transferred from parental to daughter DNA strands during post-irradiation incubation. Sedimentation in neutral sucrose gradients showed that the molecular weight of native DNA was not affected by the endonuclease treatment, suggesting that the gaps appearing in daughter strands after irradiation are not opposite dimers or that the enzyme cannot recognize dimers in the gap regions.     

Inhibition of repair of UV-damaged DNA by caffeine and mutation induction in Chinese hamster cells

The effect of caffeine on UV-irradiated Chinese hamster cells in vitro was studied on the cellular and molecular levels. Caffeine (1 mM) was shown to decrease the colony-forming ability and the frequencies of spontaneous and UV-induced mutations in Chinese hamster cells. The effect of caffeine in reducing the frequency of UV-induced mutations was demonstrated only if caffeine was present in the culture medium during the first post-irradiation cell division. Using alkaline sucrose gradient centrifugation, both parental and newly synthesized DNA in UV-irradiated and unirradiated cells were studied in the presence and absence of caffeine. Caffeine affected the sedimentation profile of DNA synthesized in UV-irradiated cells but not in unirradiated cells. Caffeine had no apparent effect on the incorporation of [³H]-thymidine into DNA of control or UV-irradiated cells, nor on the small amount of excision of UV-induced pyrimidine dimers. These results may be interpreted by a hypothesis that caffeine inhibits a certain S-phase specific, post-replication, dark-repair mechanism. The hamster and perhaps other rodent cells exposed to low doses of UV are capable of DNA replication, by-passing the non-excised pyrimidine dimers. This postulated repair process probably involves de novo DNA synthesis to seal the gaps in the nascent strand. This repair may be also responsible for the enzymatic production of mutations.     

recF-dependent and recF recB-independent DNA gap-filling repair processes transfer dimer-containing parental strands to daughter strands in Escherichia coli K-12 uvrB.

The processes for repairing DNA daughter-strand gaps were studied in UV-irradiated uvrB, uvrB recB, uvrB recF, and uvrB recB recF cells of Escherichia coli K-12. The dimer-containing parental DNA was found to be joined to daughter strands during postreplication repair in all four strains examined. Therefore, both the major (recF-dependent) and the minor (recF recB-independent) gap-filling processes repair DNA daughter-strand gaps by transferring parental strands into daughter strands.     

DNA replication and repair in a human melanoma cell-line resistant to ultra-violet-radiation.

The effect of ultra-violet (U.V.)-irradiation on DNA replication was studied in a U.V.-resistant, human melanoma cell-line (MM96). Semi-conservative synthesis of DNA was decreased about five-fold by a U.V.-dose of 100 ergs/mm2. The size of DNA fragments synthesized in irradiated cells at short times after U.V. was smaller than those synthesized in unirradiated cells. Elongation of these fragments occurred with time, and 6 hours after irradiation cells synthesized DNA in fragments of the same size as obtained in unirradiated cells. In this post-replication repair process, elongation appeared to involve de novo synthesis and was not inhibited by theophylline.     

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.     

Studies on radiation-sensitive mutants of E. coli. II. Breakage and repair of ultraviolet irradiated intracellular DNA of phage lambda

Summary It has been shown that linear DNA molecules of phage are converted to the twisted circular structure (species I) by covalent closure of the both strands at the cohesive ends after infection to the immune bacteria and that the twisted circular molecules are transformed to the circular form (species II) by a single-strand break in one of the strands of their DNA. This system offers a very sensitive method to study on the strand breaks or their repair. For characterization of the defects of ultraviolet sensitive strains, the structural changes of ultraviolet irradiated DNA in these strains were studied.Ultraviolet irradiation to phage greatly reduced the extent of conversion of the molecules to the species I in the uvrD mutant while the irradiation showed little effect on the conversion in the uvrA, B and C mutants. When infected bacteria carrying species I molecules were irradiated, the species I molecules in the uvrD mutant were disrupted while most of the molecules in the uvrA, B and C mutants kept the structure. These results indicate that in the irradiated DNA strand breaks are rarely introduced or, if introduced, repaired rapidly in the uvrA, B and C mutants and they are introduced in the uvrD mutant leading to the degradation of the DNA. These results provide a firm evidence that the defect of the uvrD mutant is different from other Her^- mutants and in the process of repair synthesis.Ultraviolet irradiation to the uvrD mutants promote the formation of the species I molecules from the infected irradiated -DNA.Such effect was not observed with the uvrA mutant. Since the uvrD mutant has UV reactivation capacity and the uvrA mutant has not, the above phenomenon is probably caused by UV reactivation and may provide a more direct method to study the mechanisms of UV reactivation than the plaque assay.Abbreviations used UV Ultraviolet light - UVr Ultraviolet light reactivation This work was aided in part by a research grant GM 08384 from the United States Public Health Service.     

Postreplication Repair of Ultraviolet Damage to DNA, DNA-Chain Elongation, and Effects of Metabolic Inhibitors in Mouse L Cells

Alkaline sucrose sedimentation studies of DNA from mouse L cells have demonstrated the following effects of several inhibitors of nucleic acid and protein synthesis on postreplication repair of ultraviolet (UV) damage to their DNA. The DNA newly synthesized by a 2 h [³H]thymidine (dThd) label following 254 nm UV irradiation of 20 J/m² is made in smaller segments of the number average mol wt (Mn) of ~10 × 10⁶ than the control of ~40 × 10⁶. The presence of caffeine at a concentration of 2 mM during the labeling of the irradiated cells reduces the Mn value to 5.8 × 10⁶, which is nearly comparable to, but somewhat larger than the expected distance between dimers in parental DNA. Afterwards, such an interrupted DNA made in the irradiated cells is completely repaired to the present maximum Mn value of 40 × 10⁶ in the consecutive 4 h chase in unlabeled dThd. The presence of the nucleic acid inhibitor, either 2 mM hydroxyurea, 50 μM arabinofuranosyl cytosine, 2 mM excess dThd or 5 μg/ml of actinomycin D (AMD) during 2- to 24-h chase periods after a 2 h postirradiation label prevents the repair to various extents, while 2 mM caffeine completely inhibits it. In the unirradiated cells, these agents except excess dThd and caffeine also interfere severely with normal elongation of nascent DNA made by a 3 min pulse label, but do not appreciably induce single chain breaks of either newly synthesized or parental DNA. The inhibition of the repair by AMD suggests that de novo elongation of DNA to close the gaps in new DNA made in the irradiated cells requires at least a template-dependent DNA polymerase. In contrast, 100 μg/ml of cycloheximide allows to complete the gap-filling repair, while it simply reduces the rates of chain growth for the repair and normal replication. Therefore, the similar sensitivity of gap-filling repair and normal replication towards the above inhibitors indicates that a preexisting DNA polymerizing system appears to be responsible and to play a common role without new protein synthesis, as far as the repair at early time after UV is concerned.     

Formation of single-stranded breaks in newly-synthesized Escherichia coli DNA following treatment with bleomycin

Changes in molecular weight of newly synthesized DNA was studied after bleomycin treatment of Escherichia coli cells. The treatment by this drug causes only the increase of dispersion in sedimentation profiles of daughter DNA strands in wild type cells. There are two alternative explanation of this fact. First, single-strand breakage does not occur in newly synthesized DNA, i.e. bleomycin-induced athyminic sites do not block cellular DNA polymerases. Second, it is possible to explain it by quick rejoining of given breaks by cell repair systems. The sedimentation profile of daughter DNA strands of recA mutant rules out the first possibility. Observed shift to low molecular weight fractions region strongly indicates the formation of single-strand breaks in newly synthesized DNA. Extensive daughter DNA degradation in xthA mutant supports the idea of the existence of very effective excision repair in the case of apyrimidinic sites. Thus, non-eliminated bleomycin-induced damage causes the formation of single-strand breaks in newly synthesized DNA strands. These breaks may be repaired in the course of recA-dependent post-replication repair.     

Bifilar enzyme-sensitive sites in ultraviolet-irradiated DNA are indicative of closely opposed cyclobutyl pyrimidine dimers.

Incubation of UV-irradiated DNA with pyrimidine dimer-DNA glycosylase in cell-free lysates prepared from Micrococcus luteus results in the appearance of double-strand breaks. It has previously been assumed that such double-strand breaks result from cleavage at closely opposed dimers. We have used hybrid molecules of bacteriophage T7 DNA comprised of two unirradiated strands, two UV-irradiated strands, or one unirradiated and one UV-irradiated strand to test this hypothesis. Bifilar cleavage was observed only with molecules consisting of two irradiated strands and no bifilar cleavage was observed after the monomerization of pyrimidine dimers by enzymatic photoreactivation. Our results indicate that at least 80% of the double-strand breaks result from cleavage at closely opposed dimers and that the induction of dimers in one strand does not influence the induction of dimers at closely opposed positions in the complementary strand of a DNA double helix.     

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.     

Molecular analysis of enhanced replication of UV-damaged simian virus 40 DNA in UV-treated mammalian cells.

Irradiation of simian virus 40 (SV40)-infected cells with low fluences of UV light (20 to 60 J/m2, inducing one to three pyrimidine dimers per SV40 genome) causes a dramatic inhibition of viral DNA replication. However, treatment of cells with UV radiation (20 J/m2) before infection with SV40 virus enhances the replication of UV-damaged viral DNA. To investigate the mechanism of this enhancement of replication, we analyzed the kinetics of synthesis and interconversion of viral replicative intermediates synthesized after UV irradiation of SV40-infected cells that had been pretreated with UV radiation. This enhancement did not appear to be due to an expansion of the size of the pool of replicative intermediates after irradiation of pretreated infected cells; the kinetics of incorporation of labeled thymidine into replicative intermediates were very similar after irradiation of infected control and pretreated cells. The major products of replication of SV40 DNA after UV irradiation at the low UV fluences used here were form II molecules with single-stranded gaps (relaxed circular intermediates). There did not appear to be a change in the proportion of these molecules synthesized when cells were pretreated with UV radiation. Thus, it is unlikely that a substantial amount of DNA synthesis occurs past pyrimidine dimers without leaving gaps. This conclusion is supported by the observation that the proportion of newly synthesized SV40 form I molecules that contain pyrimidine dimers was not increased in pretreated cells. Pulse-chase experiments suggested that there is a more efficient conversion of replicative intermediates into form I molecules in pretreated cells. This could be due to more efficient gap filling in relaxed circular intermediate molecules or to the release of blocked replication forks. Alternatively, the enhanced replication observed here may be due to an increase in the excision repair capacity of the pretreated cells.     

UV-induced repair inEscherichia coli B/r Hcr− thy trp cells: Its dependence on UV damage

Irradiation ofEscherichia coli B/r Hcr^? thy trp cells with a low UV-dose permits a post-replication repair of DNA and decreases the breakdown of DNA after a successive irradiation of cells with high UV doses. The usefulness of a repair function of the protein synthesized after a low irradiation dose increases with the increasing damage of DNA.     

Meiotic DNA Metabolism in Wild-Type and Excision-Deficient Yeast following Uv Exposure

The effects of UV irradiation on DNA metabolism during meiosis have been examined in wild-type (RAD⁺) and mitotically defined excision-defective (rad1-1) strains of Saccharomyces cerevisiae that exhibit high levels of sporulation. The rad1-1 gene product is not required for normal meiosis: DNA synthesis, RNA synthesis, size of parental and newly synthesized DNA and sporulation are comparable in RAD⁺ and rad1-1 strains. Cells were UV irradiated at the beginning of meiosis, and the fate of UV-induced pyrimidine dimers as well as changes in DNA and DNA synthesis were followed during meiosis. Excision repair of pyrimidine dimers can occur during meiosis and the RAD1 gene product is required; alternate excision pathways do not exist. Although the rate of elongation is decreased, the presence of pyrimidine dimers during meiosis in the rad1-1 strain does not block meiotic DNA synthesis suggesting a bypass mechanism. The final size of DNA is about five times the distance between pyrimidine dimers after exposure to 4 J/m². Since pyrimidine dimers induced in parental strands of rad1-1 prior to premeiotic DNA synthesis do not become associated with newly synthesized DNA, the mechanism for replicational bypass does not appear to involve a recombinational process. The absence of such association indicates that normal meiotic recombination is also suppressed by UV-induced damage in DNA; this result at the molecular level is supported by observations at the genetic level.     

Preirradiation of host cells does not alter blockage of simian virus 40 replication forks by pyrimidine dimers

Do damage-inducible responses in mammalian cells alter the interaction of lesions with replication forks? We have previously demonstrated that preirradiation of the host cell mitigates UV inhibition of SV40 DNA replication; this mitigation can be detected within the first 30 min after the test irradiation. Here we test the hypotheses that this mitigation involves either (1) rapid dimer removal, (2) rapid synthesis of daughter strands past lesions (trans-dimer synthesis), or (3) continued progression of the replication fork beyond a dimer. Cells preirradiated with UV were infected with undamaged SV40, and the effects of UV upon viral DNA synthesis were measured within the first hour after a subsequent test irradiation. In preirradiated cells, as well as in non-preirradiated cells, pyrimidine dimers block elongation of daughter strands; daughter strands grow only to a size equal to the interdimer distance along the parental strands. There is, within this first hour after UV, no evidence for trans-dimer synthesis, nor for more rapid dimer removal either in the bulk of the parental DNA or in molecules in the replication pool. Progression of the replication forks was analyzed by electron microscopy of replicating SV40 molecules. Dimers block replication-fork progression in preirradiated cells to the same extent as in non-preirradiated cells. These experiments argue strongly against the hypotheses that preirradiation of host cells results in either the rapid removal of dimers, trans-dimer synthesis, or continued replication-fork progression beyond dimers.     

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.     

The early and late modes of DNA replication in ultraviolet irradiated Syrian hamster embryo cells.

The nature of DNA replication in UV irradiated Syrian hamster embryo cells (HEC) was investigated by measuring the size distribution of nascent daughter strand DNA. During the early mode nascent strands are made in smaller pieces than in nonirradiated cells. The late mode begins when nascent strands recover to normal size. This was observed in HEC 5 h post-UV. When the late mode is operational, nascent strands elongate to parental size in greater than 2 h, whereas less than 3 h are required during early mode function. Evidence from split dose experiments demonstrates that the recovery of the size of nascent strands is not due to enhanced gap filling. Furthermore, pyrimidine dimers are probably recognized differently by the replication complex during early and late mode DNA synthesis. The late mode of replication could account for the ability of HEC to survive UV irradiation even though they are inefficient in both excision and postreplication repair.     

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.     

Repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Haemophilus influenzae

Ultraviolet-sensitive and wild-type Haemophilus influenzae cells were exposed to irradiated and unirradiated transforming deoxyribonucleic acid (DNA) containing a marker which can be linked to another marker in the cells. Lysates were made after various times of incubation and assayed for transforming activity on an excisionless recipient. Repair can be noted as an increase in activity from the irradiated donor DNA after its linkage to the recipient DNA. No repair can be observed in a mutant which is unable to integrate transforming DNA. There is a little repair in another mutant which is unable to excise pyrimidine dimers. H. influenzae cells also repair nondimer damage, as judged by the increase in activity observed in lysates made with irradiated and maximally photoreactivated DNA.     

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.