THE SYNERGISTIC ACTION OF ULTRAVIOLET AND X RADIATION ON MUTANTS OF ESCHERICHIA COLI K-12

Abstract— Prior UV irradiation increased the X-ray sensitivity of wild-type E. coli K-12. This synergistic effect of combined UV and X irradiation was also observed, but to a reduced extent, in uvrA, uvrB, uvrC , and polA mutants, but was absent in exrA, recA, recB , or recC mutants of E. coli K-12. Alkaline sucrose gradient studies demonstrated that the wand err gene-controlled, growth-medium-dependent (Type III) repair of X-ray-induced DNA single-strand breaks was inhibited by prior UV irradiation. This inhibition probably explains the synergistic effect of these two radiations on survival.     

POSTREPLICATION REPAIR IN uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 IS INHIBITED BY RICH GROWTH MEDIUM

Abstract Escherichia coli K-12 uvrA or uvrB strains grown to logarithmic phase in minimal medium showed higher survival after ultraviolet (UV) irradiation (254 nm) if plated on minimal medium (MM) instead of rich medium. This'minimal medium recovery'(MMR) was largely blocked by additional recA56 (92% inhibition) or lexA101 (77%) mutations, was partially blocked by additional recB21 (54%), uvrD3 (31%) or recF143 (22%) mutations, but additional polA1 or polA5 mutations had no effect on MMR. When incubated in MM after UV irradiation, the uvrB5 and uvrB5 uvrD3 strains showed essentially complete repair of DNA daughter-strand gaps (DSG) produced after UV radiation fluences up to ∼ 6 J/m² and ∼1 J/m², respectively, and then they accumulated unrepaired DSG as a linear function of UV radiation fluence. However, when they were incubated in rich growth medium after UV irradiation, they did not show the complete repair of DSG and unrepaired DSG accumulated as a linear function of UV radiation fluence. The fluence-dependent correlation observed for the uvrB and uvrB uvrD cells between UV radiation-induced killing and the accumulation of unrepaired DSG, indicates that the molecular basis of MMR is the partial inhibition of postreplication repair by rich growth medium. Rich growth medium can be just MM plus Casamino Acids or the 13 pure amino acids therein in order to have an adverse effect on survival, regardless of whether the cells were grown in rich medium or not before UV irradiation.     

ULTRAVIOLET INACTIVATION OF THE ABILITY OF E. COLI TO SUPPORT THE GROWTH OF PHAGE T7: AN ACTION SPECTRUM

Abstract— Ultraviolet (UV)-irradiated E. coli K-12 wild-type cells were sensitized by a post-irradiation treatment with 10^-2 M 2, 4-dinitrophenol (DNP). This effect was not seen in strains carrying a uvr mutation, suggesting that DN P interferes with the excision repair process. The polA strain was sensitized to the same extent as the wild-type strain, while the exrA strain was not affected by DNP treatment.
Recombination deficient strains ( recA, recB and recA recB ) were protected by DNP treatment after UV irradiation. This protection was abolished by the addition of a uvr mutation (i.e., in strains recA uvrB and recB uvrB ).
Alkaline sucrose gradient sedimentation studies showed that DNP treatment interfered with the rejoining of DNA single-strand breaks induced by the excision repair process. This interference was apparently specific for the exr gene-dependent branch of the uvr gene-dependent excision repair process, since the uvr and exr strains were not sensitized while the wild-type and polA strains were sensitized.     

Molecular assessment of UVC radiation-induced DNA damage repair in the stromatolitic halophilic archaeon, Halococcus hamelinensis

The halophilic archaeon Halococcus hamelinensis was isolated from living stromatolites in Shark Bay, Western Australia, that are known to be exposed to extreme conditions of salinity, desiccation, and UV radiation. Modern stromatolites are considered analogues of very early life on Earth and thus inhabitants of modern stromatolites, and Hcc. hamelinensis in particular, are excellent candidates to examine responses to high UV radiation. This organism was exposed to high dosages (up to 500 J/m(2)) of standard germicidal UVC (254 nm) radiation and overall responses such as survival, thymine-thymine cyclobutane pyrimidine dimer formation, and DNA repair have been assessed. Results show that Hcc. hamelinensis is able to survive high UVC radiation dosages and that intact cells give an increased level of DNA protection over purified DNA. The organism was screened for the bacterial-like nucleotide excision repair (NER) genes uvrA, uvrB, uvrC, as well as for the photolyase phr2 gene. All four genes were discovered and changes in the expression levels of those genes during repair in either light or dark were investigated by means of quantitative Real-Time (qRT) PCR. The data obtained and presented in this study show that the uvrA, uvrB, and uvrC genes were up-regulated during both repair conditions. The photolyase phr2 was not induced during dark repair, yet showed a 20-fold increase during repair in light conditions. The data presented is the first molecular study of different repair mechanisms in the genus Halococcus following exposure to high UVC radiation levels.     

ULTRAVIOLET RADIATION-INDUCED MUTABILITY OF uvrD3 STRAINS OF ESCHERICHIA COLI B/r AND K-12: A PROBLEM IN ANALYZING MUTAGENESIS DATA

Abstract— The involvement of the uvrD gene product in UV-induced mutagenesis in Escherichia coli was studied by comparing wild-type and uvrA or uvrB strains with their uvrD derivatives in B/r and K-12(W3110) backgrounds. Mutations per survivor (reversions to prototrophy) were compared as a function of surviving fraction and of UV fluence. While recognizing that both methods are not without problems, arguments are presented for favoring the former rather than the latter method of presenting the data when survival is less than 100%. When UV-induced mutation frequencies were plotted as a function of surviving fraction, the uvrD derivatives were less mutable than the corresponding parent strains. The B/r strains exhibited higher mutation frequencies than did the K-12(W3110) strains. A uvrB mutation increased the mutation frequency of its parental K-12 strain, but a uvrA mutation only increased the mutation frequency of its parental B/r strain at UV survivals greater than ˜ 80%. Both the uvrA and uvrB mutations increased the mutation frequencies of the uvrD strains in the B/r and K-12 backgrounds, respectively. Rather different conclusions would be drawn if mutagenesis were considered as a function of UV fluence rather than of survival, a situation that calls for further work and discussion. Ideally mutation efficiencies should be compared as a function of the number of repair events per survivor, a number that is currently unobtainable.     

ESTABLISHMENT and CHARACTERIZATION OF A MONOCLONAL ANTIBODY RECOGNIZING THE DEWAR ISOMERS OF(6–4)PHOTOPRODUCTS

Abstract— We established a monoclonal antibody(DEM–1) that recognizes UV-induced DNA damage other than cyclobutane pyrimidine dimers or(6–4)photoproducts. The binding ofDEM–1 antibody to 254 nm UV-irradiated DNA increased with subsequent exposure to UV wavelengths longer than 310 nm, whereas that of the 64M-2 antibody specific for the(6–4)photoproduct decreased with this treatment. Furthermore, the increase inDEM–1 binding was inhibited by the presence of the 64M-2 antibody during the exposure. We concluded that theDEM–1 antibody specifically recognized the Dewar photoproduct, which is the isomeric form of the(6–4)photoproduct. TheDEM–1 antibody, however, also bound to DNA irradiated with high fluences of 254 nm UV, suggesting that 254 nm UV could induce Dewar photoproducts without subsequent exposure to longer wavelengths of UV. Furthermore, an action spectral study demonstrated that 254 nm was the most efficient wavelength for Dewar photoproduct induction in the region from 254 to 365 nm, as well as cyclobutane dimers and(6–4)photoproducts, although the action spectrum values in the U V-B region were significantly higher compared with those for cyclobutane dimer and(6–4)photoproduct induction.     

ESTIMATION OF PHOTOPRODUCT FORMATION IN GERMINATING SPORES OF BACILLUS SUBTILIS

Abstract— Changes in UV sensitivity during spore germination of Bacillus subtilis mutants possessing various defects in DNA repair capacities were analysed in order to estimate the yield of the DNA photoproducts at the transient, UV resistant stage which occurs in the process of germination. It was concluded that the yield of the spore-specific photoproduct (5-thyminyl-5,6-dihydrothymine, TDHT) at the transient stage was only about 3% of that in dormant spores and the yield of the cyclobutane-type pyrimidine dimers at this stage was about 10% (or less) of that in germinated spores.     

THE BIOLOGY OF THE (6–4) PHOTOPRODUCT

The (6-4) photoproduct is an important determinant of the lethal and mutagenic effects of UV irradiation of biological systems. The removal of this lesion appears to correlate closely with the early DNA repair responses of mammalian cells, including DNA incision events, repair synthesis and removal of replication blocks. The processing of (6-4) photoproducts and cyclobutane dimers appears to be enzymatically coupled in bacteria and most mammalian cell lines examined (i.e. a mutation affecting the repair of one lesion also often affects the other), although exceptions exist in which repair capacity may be evident for one photoproduct and not the other (e.g. UV61 and the XP revertant cell line). These differences in the processing of the two photoproducts in some cell lines of human and rodent origin suggest that in mammalian cells, different pathways for the repair of (6-4) photoproducts and cyclobutane dimers may be used. This observation is further supported by pleiotropic repair phenotypes such as those observed in CHO complementation class 2 mutants (e.g., UV5, UVL-1, UVL-13, and V-H1). Indirect data, from HCR of UV irradiated reported genes and the cytotoxic responses of UV61, suggest that the (6-4) photoproduct is cytotoxic in mammalian cells and may account for 20 to 30% of the cell killing after UV irradiation of rodent cells. Cytotoxicity of the (6-4) photoproduct may be important in the etiology of sunlight-induced carcinogenesis, affecting mutagenesis as well as tumorigenesis. The intricate photochemistry of the (6-4) photoproduct, its formation and photoisomerization, is in itself extremely interesting and may also be relevant to sunlight carcinogenesis. The data reviewed in this article support the notion that the (6-4) photoproduct and its Dewar photoisomer are important cytotoxic determinants of UV light. The idea that the (6-4) photoproduct is an important component in the spectrum of UV-induced cytotoxic damage may help clarify our understanding of why rodent cells survive the effects of UV irradiation as well as human cells, without apparent cyclobutane dimer repair in the bulk of their DNA. The preferential repair of cyclobutane dimers in essential genes has been proposed to account for this observation (Bohr et al., 1985, 1986; Mellon et al., 1986). The data reviewed here suggest that understanding the repair of a prominent type of noncyclobutane dimer damage, the (6-4) photoproduct, may also be important in resolving this paradox.     

VARIATION IN THE PHOTOCHEMICAL REACTIVITY OF THYMINE IN THE DNA OF B. SUBTILIS SPORES, VEGETATIVE CELLS AND SPORES GERMINATED IN CHLORAMPHENICOL*,†

Abstract— The chief photoproduct of thymine produced in u.v. irradiated (2537Å) vegetative cells of B. subtilis is the cyclobutane-type dimer while in spores very little of this dimer is produced (maximum yield 2·6 per cent of thymine) but a new photoproduct is produced in high yield (maximum of 28·4 per cent of thymine). This difference in photochemical response appears to be due, at least in part, to a difference in uydration of the DNA. The photochemistry of thymine in isolated DNA irradiated in solution is similar to that of DNA in irradiated vegetative cells, but differs markedly from that of isolated DNA irradiated dry. The yield of cyclobutane-type thymine dimer is much reduced in isolated DNA irradiated dry but a new photoproduct of thymine. is produced which is chromatographically similar to the spore photoproduct. The yield of this photoproduct, however, is never as great as that obtained in irradiated spores. The photochemistry of the DNA thymine of spores germinated in the presence of chloramphenicol is very similar to that of normal vegetative cells. Except for hydration, the physical state of the DNA is probably not otherwise altered by germination in the presence of chloramphenicol since DNA replication is prevented by the presence of chloramphenicol. These results are also consistent with the hypothesis that the unique photochemistry of spores is due, at least in part, to the hydration state of the DNA. The acid stability of the spore photoproduct is indicated by the fact that it is isolated from irradiated spores after hydrolysis in trifluoroacetic acid at 155°C for 60 min. It still contains the methyl group of thymine as judged by the fact that for a given dose of u.v. the same yield of photoproduct was obtained whether the spores were labeled with thymine-2–C-14 or -methyl-C-14. This photoproduct is stable to reirradiation (2537Å) in solution under condiditions where thymine dimers of the cyclobutane-type are completely converted back to monomeric thymine. On a column of molecular sieve material (Sephadex-G10), the spore photoproduct elutes in a region intermediate between the cyclobutanetype thymine dimers and monomeric thymine. Of the numerous compounds tested by paper chromatography, the spore photoproduct is most similar (but not identical) in several solvents to 5–hydroxyuracil and 5–hydroxymethyluracil. Our data do not allow us to decide if the product is a monomer or a dimer. Although the photochemistry of thymine in the DNA of spores differs markedly from that for vegetative cells, several lines of evidence make it seem doubtful that the enhanced resistance of spores to u.v. relative to that of vegetative cells can be explained solely on the basis of this difference in the photochemistry of DNA thymine.     

Excision-repair capacity of UV-irradiated strains of Escherichia coli and Streptococcus pneumoniae, estimated by plasmid recovery

Although the biological role of many bacterial repair genes is known, there is still an interest in evaluating the capacity of repair pyrimidine dimers in some strains. For this purpose, we have developed a rapid assay. Cells bearing a plasmid are UV irradiated and incubated to allow recovery. The plasmid DNA is extracted, purified and treated with UV endonuclease from Micrococcus luteus that specifically produces single strand breaks at the site of pyrimidine dimers. The amount of open circular and covalently closed circular forms of the plasmid DNA after treatment and post-incubation provides an estimate of the repair capability of the host strain. The wild type strain and the uvrA mutant of Escherichia coli were used to adjust the assay. The lexA mutant of E. coli has been tested and its repair capability is equivalent to that of wild-type strain. The assay has been extended to Streptococcus pneumoniae, which is naturally deficient in photoreactivation and SOS-like functions. This strain is efficient in the repair of pyrimidine dimers, formed after UV irradiation.     

REDUCED REPAIR OF NON-DIMER PHOTOPRODUCTS IN A GENE TRANSFECTED INTO XERODERMA PIGMENTOSUM CELLS

Abstract— 4ells from patients with the sun sensitive cancer-prone disease, xeroderma pigmentosum (XP) have defective repair of UV damaged DNA with reduced excision of the major photoproduct, the cyclobutane type pyrimidine dimer. Other (non-dimer) photoproducts, have recently been implicated in UV mutagenesis. Utilizing an expression vector host cell reactivation assay, we studied UV damaged transfecting DNA that was treated by in vitro photoreactivation to reverse pyrimidine dimers while not altering other photoproducts. We found that the reduced expression of a UV damaged transfecting plasmid in XP complementation group A cells is only partially reversed by photoreactivation. E. coli photolyase treatment of pSV2catSVgpt exposed to 100 or 200 J m⁻² of 254 nm radiation removed 99% of the T4 endonuclease V sensitive sites. Transfection of XP12BE(SV40) cells with photoreactivated pSV2catSVgpt showed residual inhibition corresponding to 25 to 37% of the lethal hits to the cat gene. This residual inhibition corresponds to the fraction of non-dimer photoproducts induced by UV. This result implies that XP12BE(SV40) cells do not repair most of the non-dimer photoproducts in DNA.     

LETHALITY AND THE INDUCTION AND REPAIR OF DNA DAMAGE IN FAR, MID OR NEAR UV-IRRADIATED HUMAN FIBROBLASTS: COMPARISON OF EFFECTS IN NORMAL, XERODERMA PIGMENTOSUM AND BLOOM'S SYNDROME CELLS

Abstract Using normal human fibroblasts we have determined the ability of far (254 nm), mid (310 nm) or near (365 nm) UV radiation to: (i) induce pyrimidine dimers (detected as UV endonuclease sensitive sites) and DNA single-strand breaks (detected in alkali); (ii) elicit excision repair, monitored as unscheduled DNA synthesis (UDS); and (iii) reduce colony-forming ability. Unscheduled DNA synthesis studies were also performed on dimer excision-defective xeroderma pigmentosum (XP) cells, and the survival studies were extended to include XP and Bloom's syndrome (BS) strains. UV-induced cell killing in normal, BS and XP cells was found to relate to an equivalent dimer load per genome after 254 or 310 nm exposure, whereas at 365 nm the lethal effects of non-dimer damage appeared to predominate. Lethality could not be correlated with DNA strand breakage at any wavelength. The two XP strains examined showed the same relative UDS repair deficiency at the two shorter wavelengths in keeping with a predominant role for pyrimidine dimer repair in the expression of UDS. However, UDS was not detected in 365 nm UV-irradiated normal and XP cells despite dimer induction; this effect was due to the inhibition of DNA repair functions since 365 nm UV-irradiated normal cells showed reduced capacity to perform UDS subsequent to challenge with 254 nm UV radiation.
In short, the near UV component of sunlight apparently induces biologically important non-dimer damage in human cells and inhibits DNA repair processes, two actions which should be considered when assessing the deleterious actions of solar UV.     

NONRECIPROCAL SYNERGISTIC LETHAL INTERACTION BETWEEN 365-nm and 405-nm RADIATION IN WILD TYPE and uvrA STRAINS OF ESCHERICHIA COLI*

Abstract— Lethality by 405-nm radiation in three repair-proficient and two uvrA strains of Escherichia coli that belong to two isogenic series was greatly enhanced by prior exposures to 365-nm radiation at fluences greater than 1 times 10₆Jm_-2. Fluences at 365 nm that yielded a surviving fraction of 0.10 (>1 times 10₆ Jm_-2) in the 5 strains tested resulted in the following 405-nm fluence enhancement factors (FEF, ratio of the 405-nm F₃₇ in the absence of a prior 365-nm irradiation to that in the presence): strain K.12 AB1157 (wild type), 8.7; strain B/r (wild type), 52; strain WP2 (wild type), 25; strain WP2s (uvrA), 13; strain K.12 AB1886 (uvrA), 15. The maximal 405-nm FEF value obtained after a prior 365-nm irradiation at greater fluences was 83 in the wild-type strain B/r. Enhancement of anoxic 405-nm radiation after a prior aerobic 365-nm exposure was not detectable, suggesting that prior aerobic irradiation at 365-nm increased the effects of damage produced at 405 nm by means of an oxygen-dependent process. Single-strand breaks (or alkali-labile bonds) were produced by 405-nm radiation at 3.0 times 10_-5 breaks per 2.5 times 10₉ daltons per Jm_-2 in the polA strain P3478; pyrimidine dimers were not detected by biological assay (photoreactivation) at 405 nm. Although the introduction of different DNA lesions produced by 365- and 405-nm radiations cannot be ruled out, we propose that the strong synergistic effect of 365-nm irradiation on 405-nm lethality is the consequence of pronounced inhibition by 365-nm radiation of components of the DNA-repair systems that can mend or bypass damage produced by 405-nm radiation.     

UV mutagenic photoproducts in Escherichia coli and human cells: a molecular genetics perspective on human skin cancer

Abstract— The relevance of photoproducts produced by 254 nm irradiation to human skin cancer is first critically evaluated. Experiments identifying the mutagenic photoproducts at 254 nm are then described. Mutations are primarily due to the(6–4) photoproduct and the cyclobutane pyrimidine dimer, both in E. coli and in human cells. The(6–4) photoproduct may be more important in E. coli and the cyclobutane dimer more important in mammalian cells. In human cells, mutations occur at the C of a TC, CT, or CC cyclobutane dimer, but not at TT cyclobutane dimers, and also appear to occur, less frequently, at the C of TC and CC(6–4) photoproducts. The local structure of DNA is more important in determining the frequency of mutation at a site than is the photoproduct frequency at that site. The effect of DNA structure appears to be due to site-specific lethality.     

DNA STRAND BREAKS IN MAMMALIAN CELLS EXPOSED TO LIGHT IN THE PRESENCE OF RIBOFLAVIN AND TRYPTOPHAN

Abstract— When mammalian cells were exposed to visible-fluorescent light or near-UV light in the medium containing riboflavin and L-tryptophan, single-strand breaks appeared in their DNA. This did not occur if either riboflavin or tryptophan was omitted from the medium. The same effect was observed when cells were added to the pre-irradiated medium, indicating that a stable photoproduct was responsible. The induced DNA lesions were shown to be equally repairable in both excision proficient and defective (xeroderma pigmentosum) human cell lines. The active photoproduct formed was shown to be hydrogen peroxide. The possible relationship between these results and the near-UV induced killing of mammalian cells is discussed.     

ULTRAVIOLET LIGHT IRRADIATION OF DEFINED-SEQUENCE DNA UNDER CONDITIONS OF CHEMICAL PHOTOSENSITIZATION

Abstract— The formation of cyclobutane pyrimidine dimers and UV light-induced (6-4) products was examined under conditions of triplet state photosensitization. DNA fragments of defined sequence were irradiated with 313 nm light in the presence of either acetone qr silver ion. UV irradiation in the presence of both silver ion and acetone enhanced the formation of TT cyclobutane dimers, yet no (6-4) photoproducts were formed at appreciable levels. When photoproduct formation was also measured in pyrimidine dinucleotides, only cyclobutane dimers were formed when the dinucleotides were exposed to 313 nm light in the presence of photosensitizer. The relative distribution of each type of cyclobutane dimer formed was compared for DNA fragments that were irradiated with 254, 313, or 313 nm UV light in the presence of acetone. The dimer distribution for DNA irradiated with 254 and 313 nm UV light were very similar, whereas the distribution for DNA irradiated with 313 nm light in the presence of acetone favored TT dimers. Alkaline labile lesions at guanine sites were also seen when DNA was irradiated with 313 nm light in the presence of acetone.     

Binding of distamycin A to UV-damaged DNA

We have found that distamycin A can bind to DNA duplexes containing the (6-4) photoproduct, one of the major UV lesions in DNA, despite the changes, caused by photoproduct formation, in both the chemical structure of the base moiety and the local tertiary structure of the helix. A 20-mer duplex containing the target site, AATT.AATT, was designed, and then one of the TT sequences was changed to the (6-4) photoproduct. Distamycin binding to the photoproduct-containing duplex was detected by CD spectroscopy, whereas specific binding did not occur when the TT site was changed to a cyclobutane pyrimidine dimer, another type of UV lesion. Distamycin binding was analyzed in detail using 14-mer duplexes. Curve fitting of the CD titration data and induced CD difference spectra revealed that the binding stoichiometry changed from 1:1 to 2:1 with photoproduct formation. Melting curves of the drug-DNA complexes also supported this stoichiometry.     

SIMULTANEOUS ESTABLISHMENT OF MONOCLONAL ANTIBODIES SPECIFIC FOR EITHER CYCLOBUTANE PYRIMIDINE DIMER OR (6-4)PHOTOPRODUCT FROM THE SAME MOUSE IMMUNIZED WITH ULTRAVIOLET-IRRADIATED DNA

Six new monoclonal antibodies (TDM-2, TDM-3, 64M-2, 64M-3, 64M-4 and 64M-5) specific for ultraviolet (UV) induced DNA damage have been established. In the antibody characterization experiments, two TDM antibodies were found to show a dose-dependent binding to UV-irradiated DNA (UV-DNA), decrease of binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, binding to DNA containing cyclobutane thymine dimers, and unchanged binding to UV-DNA after photoisomerization of (6-4)photoproducts to Dewar photoproducts. These results indicated that the epitope of TDM monoclonal antibodies was the cyclobutane pyrimidine dimer in DNA. On the other hand, four 64M antibodies were found to show a dose-dependent binding to UV-DNA, unchanged binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, undetectable binding to DNA containing thymine dimers, and decrease of binding to UV-DNA after photoisomerization of (6-4)photoproducts. These results indicated that the epitope of 64M antibodies was the (6-4)photoproduct in DNA. This is the first report of the simultaneous establishment of monoclonal antibodies against the two different types of photolesions from the same mouse. By using these monoclonal antibodies, we have succeeded in measuring both cyclobutane pyrimidine dimers and (6-4)photoproducts in the DNA from human primary cells irradiated with physiological UV doses.     

Influence of Phage Proteins on Formation of Specific UV DMA Photoproducts in Phage T7

Phage T7 can be used as a biological UV dosimeter. Its reading is proportional to the inactivation rate expressed in HT7 units. To understand the influence of phage proteins on the formation of DNA UV photoproducts, cyclobutane pyrimidine dimers (CPD) and (6-4)photoproducts ((6-4)PD) were determined in T7 DNA exposed to UV radiation under different conditions: intraphage T7 DNA, isolated T7 DNA and heated phage. To investigate the effects of various wavelengths, seven different UV sources have been used. The CPD and (6-4)PD were determined by lesion-specific antibodies in an immunodot-blot assay. Both photoproducts were HT7 dose-dependently produced in all three objects by every irradiation source in the biologically relevant UV dose range (1-10 HT7). The CPD to (6-4)PD ratios increased with the increasing effective wavelength of the irradiation source and were similar in intraphage T7 DNA, isolated DNA and heated phage with all irradiation sources. However, a significant decrease in the yield of both photoproducts was detected in isolated T7 DNA and in heated phage compared to intraphage DNA, the decrease was dependent on the irradiation source. Both photoproducts were affected the same way in isolated T7 DNA and heated phage, respectively. The yield of CPD and (6-4)PD was similar in B, C-like and A conformational states of isolated T7 DNA, indicating that the conformational switch in the DNA is not the decisive factor in photoproduct formation. The most likely explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in an increased rate of dimerization and (6-4)PD production of adjacent based in intraphage T7 DNA.     

IS DNA TOPOISOMERASE INVOLVED IN THE UV EXCISION REPAIR PROCESS? NEW EVIDENCE FROM STUDIES WITH DNA INTERCALATING AND NON-INTERCALATING ANTITUMOR AGENTS

Abstract— The effects of selected DNA intercalating and non-intercalating drugs on the UV excision repair process in human fibroblasts have been examined. 9-Amino acridine, acridine orange, quinacrine, doxorubicin (adriamycin), ethidium bromide and actinomycin-D all inhibited the removal of pyrimidine dimers from cellular DNA by inhibiting the incision process as monitored by the nick translation assay and by an endonuclease-sensitive site assay. These agents also partially inhibited incision by the M. luteus endonuclease in an in vitro system. This is the only class of compounds tested to date that appears to block this early step of repair in mammalian cells. The DNA topoisomerase inhibitors, m -amsacrine and VP-16 (etoposide) and the bacterial gyrase inhibitors nalidixic acid and oxolinic acid were shown not to inhibit UV repair. As shown previously, however, novobiocin does block dimer removal and we show here that it is a potent inhibitor of the M. luteus UV endonuclease. While it has recently been demonstrated that many DNA intercalating agents block the strand-passing activity of DNA topoisomerase II giving rise to protein associated DNA strand breaks, the finding that the specific inhibitors of topoisomerase, m -AMSA and VP-16, do not inhibit repair, even though they block this strand passing activity, strongly suggests that inhibition of DNA topoisomerase is not associated with inhibition of DNA repair.