Cyclobutyl Pyrimidine Dimers Research Articles

Photoreactivation of herpes simplex virus in human fibroblasts

WHEN DNA is treated with ultraviolet light (220–300 nm) cyclobutyl pyrimidine dimers are produced between adjacent pyrimidines on a strand of DNA1. These dimers are a major cause of mutations and death in simple organisms after ultraviolet-irradiation1, and they have been implicated in the induction of skin cancer in man2,3. An important tool for assessing the deleterious effects of dimers is the photoreactivation test: the photoreactivating enzyme repairs DNA by the specific and exclusive monomerisation of dimers in a light-dependent (> 300nm) reaction4,5. If ultraviolet biological damage can be reversed by true photoenzymatic repair, then dimers have a major role in the production of that damage6. For the test to assess the role of ultraviolet-induced dimers in human carcinogenesis at least three criteria must be met: (1) human cells must possess a functional photoreactivating enzyme, (2) this enzyme must be able to monomerise dimers in DNA, and (3) the enzyme must be able to restore biological activity to ultraviolet-irradiated DNA. It has been shown that human cells meet the first two criteria7,8; we now show that human photoreactivating enzyme in fibroblasts can restore infectivity to ultraviolet-irradiated herpes simplex virus (HSV).

Purifying the Escherichia coli photoreactivating enzyme.

Purification of the photoreactivating enzyme from Escherichia coli depends on three major factors: (1) development of a suitable assay, (2) selection of a source with high enzyme levels, and (3) development of a gentle, specific purification procedure. The preparation of purified enzyme is useful both in study of DNA repair enzymes and in the analysis of the role of cyclobutyl pyrimidine dimers in the induction of biological damage by ultraviolet light.

DNA Repair in Human/Embryonic Chick Heterokaryons: Ability of Each Species to Aid the Other in the Removal of Ultraviolet-Induced Damage

Cultured human and embryonic chick fibroblasts possess different enzyme-mediated processes to repair cyclobutyl pyrimidine dimers induced in their deoxyribonucleic acid (DNA) by ultraviolet (UV) radiation. While dimers are corrected in human cells by excision repair, a photoenzymatic repair process exists in embryonic chick cells for the removal of these potentially deleterious UV photoproducts. We have utilized a sensitive enzymatic assay to monitor the disappearance, i.e. repair, of dimer-containing sites in fused populations of human and chick cells primarily consisting of multinucleate human/chick heterokaryons. Fused cultures were constructed such that UV photoproducts were present only in chick DNA when evaluating excision repair and only in human DNA when evaluating photoenzymatic repair. Based on the kinetics of site removal observed in these cultures we are led to conclude the following: Within heterokaryons per se the photoreactivating enzyme derived from chick nuclei and at least one excision-repair enzyme (presumably a UV endonuclease) derived from human nuclei act on UV-damaged DNA in foreign nuclei with an efficiency equal to that displayed toward their own nuclear DNA. Hence, after cell fusion these chick and human repair enzymes are apparently able to diffuse into foreign nuclei and once therein competently attack UV-irradiated DNA independently of its origin. In harmony with the situation in nonfused parental cultures, in heterokaryons the chick photoenzymatic repair process rapidly removed all dimer-containing sites from human DNA including the residual fraction normally acted upon slowly by the human excision-repair process.

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 ( approximately 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 approximately 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 approximately 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.

Photoreactivating enzyme from human leukocytes.

A photoreactivating enzyme has been found in human leukocytes. The specificity of the enzyme for cyclobutyl pyrimidine dimers in DNA may allow direct evaluation of the role of dimers in ultraviolet-induced carcinogenesis in man.

Substrate specificity of a bacterial UV endonuclease and the overlap with in vitro photoenzymatic repair.

Abstract— The action of an endonuclease from Micrococcus luteus, that operates on ultraviolet (UV) radiation damage, overlaps greatly with that of the yeast photoreactivating enzyme: homo and hetero cyclobutyl pyrimidine dimers in DNA are substrate for both enzymes, but pyrimidine adducts or the ‘spore photoproduct’ in DNA are not.As expected from this overlap, the action of the two enzymes is mutually interfering: single‐strand nicks introduced by the endonuclease effectively preclude photoreactivation; conversely, formation of a photoreactivating enzyme‐dimer complex can prevent nicking by the UV endonuclease. While complex formation between photoreactivating enzyme and dimers in UV‐endonuclease‐treated DNA is apparently normal, the light‐dependent repair step either fails to occur or proceeds at a very low rate. Hence, besides the requirement of a minimum chain length for the function of the photoreactivating enzyme, there is the additional restriction on the position of the dimer in a polynucleotide strand.Finally, rough approximations of the rate constants, k1 and k2, for the UV endonuclease indicate that the in vitro UV‐endonuclease‐dimer complex is relatively unstable, with dissociation of the complex being more probable than hydrolysis of the phosphodiester bond.

Excision repair (dimer excision, strand breakage and repair replication) in primary cultures of eukaryotic (bovine) cells

Excision repair of ultraviolet radiation damage to DNA in primary cultures of eukaryotic (bovine) cells can be demonstrated by the formation of DNA strand breaks after irradiation, excision of cyclobutyl pyrimidine dimers, and repair replication. Rapidly after irradiation (within 5 min) a steady state is established in which the number of strand breaks (8 000/cell at 100 ergs/ mm 2) and the rate at which bases are inserted by repair replication reach a constant level which is then maintained for several hours. Thereafter the number of strand breaks and the rate of repair replication diminish and 75 to 80% of the dimers are excised in 24 h. On the basis of the close correlation of the kinetics of the various aspects of excision repair, we have suggested that repair is effected by complexes of repair enzymes, each of which performs all steps of excision repair. Such complexes would insert between 70 and 140 bromouracil or thymine bases for each excised photoproduct and spend about 20 min for location of a photoproduct and completion of a repair region. Hydroxyurea did not interfere with rejoining of the strand breaks. Chloroquin, in contrast to reports in E. coli, had no specific effect on excision repair. Immediately after irradiation, cells exposed to high pH and a chelating agent (EDTA) lyse to release DNA more rapidly than control cells. This accelerated release, which may be the consequence of DNA strand breaks or UV damage to membranes and proteins, is lost by about 3 h after irradiation, suggestive of a recovery process distinct from excision repair.

Studies on the mechanism of the photosensitized dimerization of pyrimidines.

Thymine-2-14C was irradiated with UV light (>300 nm) in water solutions in the presence of different sensitizers. Pyrimidines upon irradiation with wavelengths of 300—320 nm in the presence of some ketones as sensitizers, yield cyclobutyl pyrimidine dimers. The reaction occurs through a triplet-triplet energy transfer from the sensitizer to the substrate. The energy transfer, however, does not occur through a simple physical mechanism. The ability of a donor (e. g. ketone molecule) in its excited state to form a complex with the acceptor (pyrimidine molecule) appears to be a prerequisite in this type of photodimerization reaction. A correlation of the photochemical behavior of different ketones in this reaction with the nature of their excited states has been proposed. Carbonyl compounds possessing n, π* lowest triples are active in inducing pyrimidine dimerizations. By introducing some different electron donating groups to the ketone molecules that lower its π, π* state below its n, π* level, the sensitizing ability of the molecule appears to decrease or disappears completely. Ethylacetoacetate was found to be a more effective sensitizer than acetone or acetophenone. Likewise, dihydroxyacetone was found to be a potent photosensitizer in dimerization of thymine. Urocanic acid (UCA), a major UV absorbing compound in mammalian skin, did not show any sensitizing ability to induce cyclobutyl pyrimidine dimers on irradiation in the presence of thymine, with wavelengths greater than 300 nm.

Repair of pyrimidine dimer damage induced in yeast by ultraviolet light.

Crude extracts from ultraviolet (UV)-irradiated yeast cells compete with UV-irradiated transforming deoxyribonucleic acid (DNA) for photoreactivating enzyme. The amount of competition is taken as a measure of the level of cyclobutyl pyrimidine dimers in the yeast DNA. A calibration of the competition using UV-irradiated calf thymus DNA indicates that an incident UV dose (1,500 ergs/mm(2)) yielding 1% survivors of wild-type cells produces between 2.5 x 10(4) to 5 x 10(4) dimers per cell. Wild-type cells irradiated in the exponential phase of growth remove or alter more than 90% of the dimers within 220 min after irradiation. Pyrimidine dimers induced in stationary-phase wild-type cells appear to remain in the DNA; however, with incubation, they become less photoreactivable in vivo, although remaining photoreactivable in vitro. In contrast, exponentially growing or stationary-phase UV-sensitive cells (rad2-17) show almost no detectable alteration of dimers. We conclude that the UV-sensitive cells lack an early step in the repair of UV-induced pyrimidine dimers.

Formation of thymine dimers in mammalian skin by ultraviolet radiation in vivo.

Abstract— Ultraviolet radiation of 220–300 nm is known to produce cyclobutyl pyrimidine dimers in extracellular DNA, in bacteria, and in mammalian cells in culture. The formation in vivo of such dimers in mammalian skin has remained inferential. We report that one of the important and recognizable biologic events that occurs in mammalian skin during irradiation is the formation of thymine dimers. [3H]‐labelled thymidine was applied to the epilated skin of guinea pigs to label their DNA. Animals were irradiated individually, using wavelengths of either 254, 285–350, or 320–400 nm. Immediately after irradiation, epidermis was separated from the rest of the skin and homogenized; DNA and RNA were isolated. Irradiation with wavelengths of 285–350 nm, which included the sunburn‐producing spectrum (i.e., 290–320 nm), produced thymine dimers (1·7–2·6 per cent of the total [3H]‐thymine incorporated into DNA). Irradiation with 254nm also produced fewer dimers (0·46–1·2 percent); and 320–400 nm produced none. The dimer could be cleaved by 250 nm radiation to form thymine. The epidermal cell damage by ultraviolet radiation, particularly by the sunburn‐producing spectrum (290–320 nm), may be related to the formation of such dimers.

Inhibition of Pyrimidine Dimer Formation in DNA by Cationic Molecules: Role of Energy Transfer

In addition to the acridine dyes, acridine orange and proflavine, we find that three other cationic molecules which bind to DNA-ethidium bromide, chloroquine, and methyl green-inhibit the production of cyclobutyl pyrimidine dimers by ultraviolet radiation. Intercalation is not necessary for dimer inhibition. The long range nature of the inhibition implies that energy transfer is responsible. The transfer is between the lowest excited singlet state of DNA and the acceptor singlet, and seems to involve the Förster mechanism.

Mechanisms of Inhibition of Pyrimidine Dimer Formation in Deoxyribonucleic Acid by Acridine Dyes

The ultraviolet (UV)-induced formation of cyclobutyl pyrimidine dimers in Escherichia coli deoxyribonucleic acid (DNA) in vitro has been investigated in terms of the mechanism of inhibition by acridine dyes, the effect on dimer yield of specific singlet and triplet quenchers, and the mechanism of dimer formation. Our results indicate that (a) energy transfer is important in dimer reduction by acridines, (b) this transfer occurs from the singlet (S(1)) of DNA, and (c) at room temperature triplet quenchers do not reduce dimer yield in DNA.