UV Lesions In DNA Research Articles

Structural analysis of DNA containing (6-4) photoproduct in complex with distamycin A by NMR

We reported that distamycin A can bind to DNA containing the (6-4) photoproduct, one of the major UV lesions in DNA, by means of CD spectroscopy. Here, we have analyzed the structure of DNA containing the (6-4) photoproduct in complex with distamycin A by NMR. Broadening of NMR resonances and large chemical shift perturbation have been observed for residues located close to the (6-4) photoproduct in DNA, indicating the binding to this region. Intermolecular NOEs between DNA and distamycin A have been identified for some of these residues, suggesting the formation of the relatively tight complex. On the basis of these NOEs, structure determination of the complex is now in progress.

Complex Formation with Damage Recognition Protein Rad14 Is Essential for Saccharomyces cerevisiae Rad1-Rad10 Nuclease To Perform Its Function in Nucleotide Excision Repair In Vivo

Nucleotide excision repair (NER) in eukaryotes requires the assembly of a large number of protein factors at the lesion site which then coordinate the dual incision of the damaged DNA strand. However, the manner by which the different protein factors are assembled at the lesion site has remained unclear. Previously, we have shown that in the yeast Saccharomyces cerevisiae, NER proteins exist as components of different protein subassemblies: the Rad1-Rad10 nuclease, for example, forms a tight complex with the damage recognition protein Rad14, and the complex of Rad1-Rad10-Rad14 can be purified intact from yeast cells. As the Rad1-Rad10 nuclease shows no specificity for binding UV lesions in DNA, association with Rad14 could provide an effective means for the targeting of Rad1-Rad10 nuclease to damage sites in vivo. To test the validity of this idea, here we identify two rad1 mutations that render yeast cells as UV sensitive as the rad1Delta mutation but which have no effect on the recombination function of Rad1. From our genetic and biochemical studies with these rad1 mutations, we conclude that the ability of Rad1-Rad10 nuclease to associate in a complex with Rad14 is paramount for the targeting of this nuclease to lesion sites in vivo. We discuss the implications of these observations for the means by which the different NER proteins are assembled at the lesion site.

Replication of damaged DNA by translesion synthesis in human cells

Most types of DNA damage block the passage of the replication machinery. In order to bypass these blocks, cells employ special translesion synthesis (TLS) DNA polymerases, which have lower stringency than replicative polymerases. DNA polymerase η is the major polymerase responsible for bypassing UV lesions in DNA and its absence results in the variant form of the genetic disorder, xeroderma pigmentosum. Other TLS polymerases have specificities for different types of damage, but their precise roles inside the cell have not yet been established. These polymerases are located in replication factories during DNA replication and the polymerase sliding clamp PCNA plays an important role in mediating switching between different polymerases.

Analysis of distamycin A binding 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, in spite of the changes caused by photoproduct formation in the chemical structure of the base moiety and the local tertiary structure of the duplex. 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 the photoproduct formation. Melting curves of the drug-DNA complexes also supported this stoichiometry.

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.

Two-Wavelength Fluorescence Assay for DNA Repair

A simple and reliable quantitative assay for measuring cellular DNA repair capacity has been developed. It is based on the host cell reactivation of the UV-irradiated plasmid pEGFP carrying the marker gene for the enhanced green fluorescent protein (EGFP). As a reference we used the plasmid pEYFP carrying the gene for a red-shifted fluorescent protein (EYFP). Both proteins can be excited by visible light with a maximum at 488 nm, but EGFP emits with a maximum at 509 nm, while EYFP emits with a maximum at 527 nm. This makes it possible to monitor the expression of the two genes simultaneously by measuring the fluorescence at two wavelengths. HEK293 cells were cotransfected with a mixture of UV-irradiated pEGFP and undamaged pEYFP. At different time intervals after transfection the fluorescence of EGFP was determined relative to the fluorescence of EYFP to compensate for any differences in the transfection efficiency or other experimental variables. It was used to calculate the number of UV lesions in DNA and hence the repair capacity of the host cells. It was found that HEK293 cells were able to repair approximately 1.4 UV lesions per 1000 nucleotides DNA for 12 h on the average.

Preferential Binding of High Mobility Group 1 Protein to UV-damaged DNA: ROLE OF THE COOH-TERMINAL DOMAIN

Binding of chromosomal high mobility group 1 protein (HMG1) to UV-damaged DNA has been studied with oligonucleotides containing a single dipyrimidine site for formation of UV photolesions. Irradiation of an oligonucleotide with unique TT dinucleotide resulted in generation of cyclobutane pyrimidine dimer with no evidence for induction of (6-4) photoproducts, whereas the analysis of irradiated TC-containing oligonucleotide detected (6-4) photoproducts but not cyclobutane pyrimidine dimers. Mobility shift assays have revealed that HMG1 protein binds preferentially to irradiated TT and TC oligonucleotides. Photoreversal of cyclobutane pyrimidine dimers with DNA photolyase and hydrolysis of the (6-4) photoproducts with hot alkali substantially reduced but did not eliminate binding of HMG1. The protein, therefore, appears to bind the two main types of UV damages in DNA, but some other photolesion(s) contributes to the preferential binding of HMG1 to irradiated DNA. By quantifying gel shift assays and considering the efficiencies of lesion formation, we determined dissociation constants of 1.2 +/- 0.5 and 4.0 +/- 1.5 microM for irradiated TT and TC oligonucleotides, respectively, and 70 +/- 20 microM for the control non-irradiated probes. Tryptic removal of the acidic COOH-terminal domain of HMG1 significantly affected binding of the protein to both irradiated and intact oligonucleotides. The potential role of HMG1 in recognition of the UV lesions in DNA is discussed.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes XII. Evidence for enhanced photolysis of enzyme—substrate complexes by a 2-photon reaction

Yeast photoreactivating enzyme (PRE), preilluminated with wavelengths ranging from the near-UV to the red spectral region, forms with 254 nm-irradiated transforming DNA of Haemophilus influenzae enzyme—substrate complexes that are more efficiently photorepaired than complexes formed from non-preilluminated PRE. The action spectrum for this “preillumination effect”, previously shown to have a maximum in the near-UV region, has another maximum near 577 nm. In complexes formed from non-preilluminated PRE the repair probability per incident photon is only about 25% of that in complexes formed from preilluminated PRE, if low-intensity photoreactivating light is applied continuously or as a sequence of flashes. However, photoreactivating light in the form of a single, high-intensity flash of 1 msec duration raises the repair probability to >50%. Two light flashes, discharged with a delay of slightly more than a millisecond, may already achieve less photorepair than the same energy given as a single flash. These results are explained by the assumption that the great majority of PRE molecules in a non-preilluminated preparation have reduced activity (of the order of 1 4 of maximal activity). These less reactive molecules form enzyme—substrate complexes (“non-activated complexes”) in which the repair probability per incident photon is considerably increased if 2 or more photons are absorbed within a time period of the order of milliseconds. This phenomenon, tentatively termed “2-photon photolysis” does not occur in “activated complexes” (i.e. those formed from preilluminated enzyme). The data are compatible with the suggestion that the first absorption leads to a metastable excited state of the complex, during which the repair probability is increased by absorption of another photon. The generally observed heterogeneity of the photolytic response of enzyme—substrate complexes can be partly explained by heterogeneity of PRE molecules regarding their activity. In particular, uncontrolled exposure of the enzyme to almost any kind of room light before its experimental use can enhance the heterogeneity.

Analysis of photoernzymatic repair of UV lesions in DNA by single light flashes. XI. Light-induced activation of the yeast photoreactivating enzyme

Photoreactivating enzyme (PRE) from yeast (as semi-crude extract, or in highly purified form) shows increased activity if its is illuminated with near UV or short wavelength visible light prior to its use for photoenzymatic repair of UV-induced pyrimidine dimers in transforming DNA in vitro. This effect results from an alternation in PRE molecules changing those with low activity in the light-dependent step of the reaction to a higher activity. Light-induced activation of PRE preparations is slowly lost by dark storage for several hours to 1 day (faster at 23°C than at 5°C), but can be recovered repeatedly by renewed preillumination. The action spectrum for these preillumination effects generally resembles that for the photoenzymatic repair reaction itself, having its maximum in the same 355–385 nm region as the latter, but light of somewhat longer wavelengths (546 nm) is still effective. Preilluminated PRE is also more stable to thermal inactivation (65°C) than untreated enzyme.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes. X. Stability of the photoreactivating enzyme in resting Escherichia coli cells

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes. X. Stability of the photoreactivating enzyme in resting Escherichia coli cells

Transcription unit mapping in bacteriophage T7. I. In vivo transcription by Escherichia coli RNA polymerase.

Premature termination of transcription at UV lesions in DNA permits a study of the sequential order of transcription by E. coli RNA polymerase of the genes in the early region of T7. This analysis is extended to the transcription by E. coli polymerase of the late region of T7 by deleting the early terminator. The results demonstrate the existence of a single large transcription unit spanning the early region, with a promotor located at the left end of the T7 genome. Furthermore, there are no initiation sites for E. coli RNA polymerase in the late region. When E. coli polymerase transcribes the late region, it does so exclusively by initiation at the early promotor.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes IX. Excess production of photoreactivating enzyme in E. Coli B s-1-160 under different growth conditions, and its suppression by adenine

Summary The extent of photoenzymatic repair resulting from a single light flash is determined by the number of photoreactivating enzyme (PRE) molecules present within cells. This permitted us to study the dependence of PRE production on growth conditions in E. coli B s-1 -160 (an adenine-requiring mutant of strain B s-1 with about a 6-fold increased PRE content), and to compare it with that of the parental strain B s-1 . The excess PRE production was observed only in B s-1 -160 cells entering the stationary phase; their PRE content during exponential growth differed little from that in B s-1 . Supplementation of broth with adenine at concentrations from 40 to 200 μg/ml progressively decreases the amount of PRE present in stationary phase B s-1 -160 cells, but does not affect the PRE content of B s-1 cells. The same effect was observed with several adenine analogues and related substances, some of them unable to support growth of B s-1 -160 in minimal media. The results indicate that excess PRE production occurs only under conditions of severe adenine shortage, i.e. when growth of adenine-requiring cells comes to a halt due to exhaustion of the adenine supply. This suggests that adenine or some related compound is involved in a regulatory mechanism responsible for the low level of PRE production that is characteristic of E. coli .

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes V. Determination of the reaction-rate constants in E. coli cells

It was shown earlier that the use of intense light flashes permits analyzing in greater detail the reaction steps involved in photoenzymatic repair (PER). This paper describes experiments in which the reaction-rate constants for the formation of enzyme-substrate complexes ( k 1) and dark dissociation of the complexes ( k 2), and the photolytic constant ( k p ) for the light-dependent repair step have been determined in E. coli B s−1. All three reactions are heterogeneous with respect to these constants. Presumably this reflects the diversity of pyrimidine dimers in irradiated DNA, all of which serve as substrate for photoreactivating enzyme (PRE); the following values are weighted averages. At room temperature, k 1 equals 1.8·10 −3 cell volumes·molecules −1·sec −1, which for a cell volume of 10 −15 liter corresponds to 1.1·10 6 liter·mole −1·sec −1. For k 2, values ranging from 1.9·10 −3 to 1.3·10 −2 sec −1 have been obtained in different experimental approaches, depending on which criteria were used. k p has been determined under conditions where all of the substrate is complexed with photoreactivating enzyme at the beginning of the illumination. For the wavelengths 385, 366, and 355 nm k p values of, respectively, 1.75·10 −3, 1.37·10 −3, and 1.13·10 −3 mm 2·erg −1, have been obtained. From the values the product of ϵ (the molar extinction coefficient of the enzyme-substrate complexes) and Φ (the quantum yield of the photolytic reaction) can be calculated. At the most effective wavelength (385 nm), ϵΦ equals 2.4·10 4 liter·mole −1·cm −1, indicating that both ϵ and Φ must be high. ϵ must be ⩾2.4·100 4 lliter·mole −1·cm −1, since Φ cannot exceed 1; Φ is probably between 10 −1 and 1, since ϵ values >10 5 are unlikely. k 1 and k 2 show positive temperature dependence in the range 5 to 37°, corresponding to activation energies of 11 kcal·mole −1 and about 4.5 kcal·mole −1 for the respective steps, while k p was earlier shown to be essentially temperature-independent in this range.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes VI. Rate constants for enzyme-substrate binding in vitro between yeast photoreactivating enzyme and ultraviolet lesions in Haemophilus transforming DNA

Rate constants k 1 for formation, and k 2 for dissociation of the enzyme-substrate complexes of yeast photoreactivating enzyme and ultraviolet lesions in Haempphilus influenzae transforming DNA, have been measured as functions of temperature, pH and ionic strength in vitro. The measurements employed intense light flashes which cause repair of essentially all lesions complexed, to determine the number of complexes existing at any moment. Absolute concentrations of substrate were referred to the known numbers of pyrimidine dimers in irradiated DNA, while enzyme concentrations were determined by titration against substrate in a concentration range where the binding was essentially complete. The results reported permit a choice of optimum conditions for photoreactivation under various conditions of illumination. UV lesions are heterogeneous with respect to both formation and dissociation of complexes, and the values determined for the constants are weighted averages. The two rate constants depend on temperature as described by the Arrhenius expression with activation energies of about 9.3 and 5.5 kcal·mole −1, respectively. This particular difference between the two activation energies indicates that the binding energy in the complex is small. Both constants increase with pH over the range 6.0–7.7, but their ratio, which governs the equilibrium complexing in the dark, is higher at the lower pH. k 1 depends critically on the salt concentration, the position of the sharp maximum varying with the particular ions present, while k 2 increases generally with salt concentration over the range studied. Simple empirical expressions have been found which quantitatively describe the behavior of both constants as a function of ionic strength.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes VII. Photolysis of enzyme-substrate complexes in vitro

The extent of complexing of the repairable ultraviolet lesions in Haemophilus influenzae transforming DNA with yeast photoreactivating enzyme in the dark can be determined by applying intense light flashes, which cause repair of essentially all lesions complexed. With full complexing of repairable lesions the kinetics of photoreactivation by steady illumination reflect only the photochemical reaction occurring in the enzyme-substrate complex. Complexes are heterogeneous with respect to their rates of photolysis, described by the first-order rate constant k 3. At early stages of repair k 3 = k p I, where I is the light intensity and k p is the photolysis constant. The measured value of k p permits calculation of the product ϵΦ, where ϵ is the molar extinction coefficient for the complex, and Φ is the quantum yield for photolysis, giving an absolute action spectrum. In the region of most effective wavelengths (355–385 nm), ϵ > 10 4 liter·mole −1·cm −1, and Φ > 0.1, with the possibility that Φ ∼ 1. k p is independent of temperature and ionic strength over a range producing large effects on the rate of complex formation, but does increase with pH over the range 6.7–7.4. For the slower repair of a minority of lesions the measured value of k p depends somewhat on intensity and continuity of illumination, suggesting some differences not yet understood. At the usual illumination intensities used for photoreactivation k 3 is greater than k 2 the rate constant for dark dissociation of complexes.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes VIII. Inhibition of photoenzymatic repair of UV lesions in E. coli DNA by caffeine

The rate of photoenzymatic repair (PER) obtained with continuous illumination of UV-irradiated E. coli B s−1 cells is much decreased in the presence of 16 mg/ml caffeine. This inhibitory effect is usually not observed in B/r cells. It occurs in T1 phage infecting either B s−1 or B/r, indicating that its absence in B/r cells has not a trivial explanation such as destruction or decreased uptake of the inhibitor. Experimental evidence is presented that caffeine interferes strongly with the formation of enzyme-substrate (ES) complexes, presumably by binding to DNA at, or close to, the repairable photoproducts. This results in a lower equilibrium of complex formation in the dark. A single caffeine molecule can block the formation of a complex; this effect is fully reversible when the caffeine is diluted out. Caffeine does not interfere with the photolysis of existing ES complexes. Failure to observe, under usual conditions, caffeine inhibition of PER in irradiated B/r cells is explained by the large number of UV lesions in these cells, compared to much smaller numbers in B s−1 cells or in phage infecting either type of cell. Due to this high number, complex formation in B/r cells is so rapid that—in spite of the slowdown by caffeine—most of the PRE is tied up in ES complexes in the steady state during continuous illumination at moderate intensity. Only at very high light intensity, where the steady-state concentration of complexes is lower, is the caffeine inhibition observed. Correspondingly, illumination of irradiated B s−1 cells at very low light intensity results in decreased caffeine inhibition of PER.

Analysis of photoenzymatic repair of UV lesions in DNA by single flashes IV. Mutations affecting the number of photoreactivating enzyme molecules in E. coli cells

Analysis of photoenzymatic repair of UV lesions in DNA by single flashes IV. Mutations affecting the number of photoreactivating enzyme molecules in E. coli cells

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes: III. Comparison of the repair effects at various temperatures between +37° and −196°

UV-irradiated transforming DNA of Haemophilus influenzae can be repaired in vitro in the presence of yeast photoreactivating enzyme (PRE) by an intense light flash of about 1 msec duration. Such a flash repaires a fraction > 0.9 of enzyme-complexed photorepairable lesions at all temperatures between +37° and −40°, but only a fraction of about 0.25 or 0.1 at −78° or −196° respectively. A similar decline below −40° is observed for photoenzymatic repair (PR) of E. coli B s−1 cells and for PR of transforming DNA by continuous illumination with white light. The decreased effects at low temperatures result neither from damage to the reaction mixture by freezing or by the illumination itself, nor do they result from dissociation of the PRE-substrate complexes in the dark. The drastic reduction in the extent of PR is observed already below −2° if the photolytic reaction is limited by using a less intense light flash. These results are best explained by assuming that the relative repair efficiency of incident photons drops substantially below −2°, but this is not noticed with a very intense flash, where the number of incident photons is excessive. It remains open whether the decreasing repair efficiency with decreasing temperature reflects reduced absorbance or a reduced quantum yield of the absorbed photons. Increased PR effects at −78° and −196° can be obtained by sequential flashes or by increased periods of illumination with white light, but the kinetics indicate the presence of 2 or more fractions of PRE-substrate complexes, differing in their probabilities of being repaired. At low temperature as well as at room temperature flashes photolyse a majority of the complexes more efficiently than equivalent doses of continuous illumination, suggesting an enhancement of PR by absorption of 2 or more photons within a limited time interval.

ULTRAVIOLET LIGHT ALTERATION OF CELLULAR DEOXYRIBONUCLEIC ACID in vivo

DNA after irradiation with ultraviolet light was immunogenic in rabbits and elicited serum antibodies reacting specifically with UV-irradiated DNA. The serological reactions were demonstrated by immunodiffusion, complement fixation, and immunofluorescence. By immunofluorescence, antisera reacted with cell nuclei of irradiated tissue sections but not with unirradiated tissue. This method was employed to show the presence of UV lesions in tissues of mice exposed to UV light. UV lesions in DNA were present in nuclei of epidermal cells, and in heavily irradiated animals they were also detected in the corium immediately below the epidermis. The method is useful not only for directly demonstrating UV lesions of DNA but also for localizing such lesions in tissues.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes: II. In vivo studies with Escherichia coli cells and bacteriophage

Intracellular complexes of UV lesions in DNA with molecules of photoreactivating enzyme (PRE) in E. coli can be photolysed with a probability close to 1 by single light flashes of about 1 millisecond duration. The observed photoreactivation (PR) effect permits the number of PRE-substrate complexes present at the time of the flash to be determined on the following basis: ( a) The number of PRE-substrate complexes equals the number of lesions repaired; ( b) The number of photorepairable UV lesions present in DNA equals the number of pyrimidine dimers recoverable from acid hydrolysates; a UV dose of 1 erg·mm −2 of 2537 Å radiation causes the formation of approximately 6.5 dimers per E. coli chromosome; ( c) The observed PR effect can be expressed by the formal “dose-reduction”, i.e. by the reduction in the number of dimers. The kinetics of intracellular complex formation can be followed by varying the time interval between UV irradiation and flash reactivation. Stationary phase B s−1 cells, irradiated with 4.8 erg·mm −2 form a maximum number of complexes within approximately 5 min at room temperature, 50% of them being formed within the first 10–15 sec. For greater UV doses (6.4–24 erg·mm −2) the maximum number formed reaches a constant limiting value of 20, indicating that this is approximately the number of PRE molecules in these cells. Experiments with sequential flashes at various temperatures between 2° and 44° show that both the maximum extent of complex formation and the photolytic rate constant are temperature-independent in this range. Hence, the usually observed temperature-dependence of PR under conditions of continuous illumination reflects the temperature-dependence of the complex formation only. Extensive PR effects with single light flashes were also found in UV-irradiated phage T1 after injection of unirradiated B s−1 cells. The effects are much smaller in irradiated host cells due to competition by the bacterial DNA. Creating the competitive substrate after the phage DNA has reacted with the PRE results in a time-dependent decrease of the PR effect in phage as the enzyme equilibrates between the host-cell and phage DNA's. The slow rate of equilibration indicates a relatively high stability of the complexes in the dark. A heterogeneity of the photoproducts is evident with regard to both complex formation and stability. Comparative experiments with phage infecting other host strains indicate that the number of PRE molecules in strain B/r equals that in B s−1, but is lower in the K12 derivatives AB 1157, AB 2437 and AB 2480.