Micrococcus Luteus UV Endonuclease Research Articles

Multiphoton Near‐Infrared Femtosecond Laser Pulse‐Induced DNA Damage With and Without the Photosensitizer Proflavine

Abstract— The excitation of pBR322 supercoiled plasmid DNA with intense near‐IR 810 nm fs laser pulses by a simultaneous multiphoton absorption mechanism results in single‐strand breaks after treatment of the irradiated samples with Micrococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of three (or more) 810 nm IR photons (pulse width ∼140 fs, 76 MHz pulse repetition rate, average power output focused through 10×microscope objective is ∼1.2 MW/cm2). Direct single‐strand breaks (without treatment with M. luteus) were not observed under these conditions. However, in the presence of 6 μM of the intercalator proflavine (PF), both direct single‐and double‐strand breaks are observed under conditions where substantial fractions of undamaged supercoiled DNA molecules are still present. The fraction of direct double‐strand breaks is 30 ± 5% of all measurable strand cleavage events, is independent of dosage (up to 6.4 GJ/ cm2) and is proportional to In, where I is the average power/area of the 810 nm fs laser pulses, and n = 3 ± 1. The nicking of two DNA strands in the immediate vicinity of the excited PF molecules gives rise to this double‐strand cleavage. In contrast, excitation of the same samples under low‐power, single‐photon absorption conditions (∼400–500 nm) gives rise predominantly to single‐strand breaks, but some double‐strand breaks are observed at the higher dosages. Thus, single‐photon excitation with 400–500 nm light and multiphoton activation of PF by near‐IR fs laser pulses produces different distributions of single‐and double‐strand breaks. These results suggest that DNA strand cleavage originates from unrelaxed, higher excited states when PF is excited by simultaneous IR multiphoton absorption processes.

Multiphoton near-infrared femtosecond laser pulse-induced DNA damage with and without the photosensitizer proflavine.

The excitation of pBr322 supercoiled plasmid DNA with intense near-IR 810 nm fs laser pulses by a simultaneous multiphoton absorption mechanism results in single-strand breaks after treatment of the irradiated samples with Micrococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of three (or more) 810 nm IR photons (pulse width approximately 140 fs, 76 MHz pulse repetition, average power output focused through 10x microscope objective is approximately 1.2 MW/cm2). Direct single-strand breaks (without treatment with M. luteus) were not observed under these conditions. However, in the presence of 6 microM of the intercalator proflavine (PF), both direct single- and double-strand breaks are observed under conditions where substantial fractions of undamaged supercoiled DNA molecules are still present. The fraction of direct double-strand breaks is 30 +/- 5% of all measurable strand cleavage events, is independent of dosage (up to 6.4 GJ/cm2) and is proportional to In, where I is the average power/area of the 810 nm fs laser pulses, and n = 3 +/- 1. The nicking of two DNA strands in the immediate vicinity of the excited PF molecules gives rise to this double-strand cleavage. In contrast, excitation of the same samples under low-power, single-photon absorption conditions (approximately 400-500 nm) gives rise predominantly to single-strand breaks, but some double-strand breaks are observed at the higher dosages. Thus, single-photon excitation with 400-500 nm light and multiphoton activation of PF by near-IR fs laser pulses produces different distributions of single- and double-strand breaks. These results suggest that DNA strand cleavage originates from unrelaxed, higher excited states when PF is excited by simultaneous IR multiphoton absorption processes.

The Topodynamics of Incision of UV-irradiated Covalently Closed DNA by the Escherichia coli Uvr(A)BC Endonuclease

The Escherichia coli Uvr(A)BC endonuclease (Uvr(A)BC) initiates nucleotide excision repair of a large variety of DNA damages. The damage recognition and incision steps by the Uvr(A)BC is a complex process utilizing an ATP-dependent DNA helix-tracking activity associated with the UvrA2B1 complex. The latter activity leads to the generation of highly positively supercoiled DNA in the presence of E. coli topoisomerase I in vitro. Such highly positively supercoiled DNA, containing ultraviolet irradiation-induced photoproducts (uvDNA), is resistant to the incision by Uvr(A)BC, whereas the negatively supercoiled and relaxed forms of the uvDNA are effectively incised. The E. coli gyrase can contribute to the above reaction by abolishing the accumulation of highly positively supercoiled uvDNA thereby restoring Uvr(A)BC-catalyzed incision. Eukaryotic (calf thymus) topoisomerase I is able to substitute for gyrase in restoring this Uvr(A)BC-mediated incision reaction. The inability of Uvr(A)BC to incise highly positively supercoiled uvDNA results from the failure of the formation of UvrAB-dependent obligatory intermediates associated with the DNA conformational change. In contrast to Uvr(A)BC, the Micrococcus luteus UV endonuclease efficiently incises uvDNA regardless of its topological state. The in vitro topodynamic system proposed in this study may provide a simple model for studying a topological aspect of nucleotide excision repair and its interaction with other DNA topology-related processes in E. coli.

Nucleotide level detection of cyclobutane pyrimidine dimers using oligonucleotides and magnetic beads to facilitate labelling of DNA fragments incised at the dimers and chemical sequencing reference ladders.

We present a method for detecting cyclobutane pyrimidine dimers (CPDs) at the nucleotide level and an adaptation of Maxam-Gilbert sequencing for generating sequence reference ladders. UV irradiated genomic DNA from Escherichia coli was digested with restriction enzyme(s) and incised at the CPDs with Micrococcus luteus UV endonuclease. The subsequent specific fragments were separated using a biotin labelled oligonucleotide containing a sequence complementary to the fragments of interest and streptavidin magnetic beads. These fragments were then radiolabelled on the beads just prior to the running of the sequencing gel. For generating sequence reference ladders, the unlabelled DNA fragments of interest were base-specifically modified and subsequently cleaved at the A+G or C+T sites using the rapid Maxam-Gilbert sequencing treatments. These chemically cleaved fragments can be stored almost indefinitely. Whenever the sequence reference ladders are required, the chemically cleaved fragments can be labelled alongside the CPD-specifically incised DNA fragments using the same procedure. The adaptation of the method to detect other types of DNA damage is also discussed.

Purification and cloning of Micrococcus luteus ultraviolet endonuclease, an N-glycosylase/abasic lyase that proceeds via an imino enzyme-DNA intermediate.

Although Micrococcus luteus UV endonuclease has been reported to be an 18-kDa enzyme with possible homology to the 16-kDa endonuclease V from bacteriophage T4 (Gordon, L. K., and Haseltine, W. A. (1980) J. Biol. Chem. 255, 12047-12050; Grafstrom, R. H., Park, L., and Grossman, L. (1982) J. Biol. Chem. 257, 13465-13474), this study describes three independent purification schemes in which M. luteus UV damage-specific or pyrimidine dimer-specific nicking activity was associated with two proteins of apparent molecular masses of 31 and 32 kDa. An 18-kDa contaminant copurified with the doublet through many of the chromatographic steps, but it was determined to be a homolog of Escherichia coli ribosomal protein L6. Edman degradation analyses of the active proteins yielded identical NH2-terminal amino acid sequences. The corresponding gene (pdg, pyrimidine dimer glycosylase) was cloned. The protein bears strong sequence similarities to the E. coli repair proteins endonuclease III and MutY. Nonetheless, traditionally purified M. luteus protein acted exclusively on cis-syn thymine dimers; it was unable to cleave site-specific oligonucleotide substrates containing a trans-syn -I, (6-4), or Dewar thymine dimer, a 5,6-dihydrouracil lesion, or an A:G or A:C mismatch. The UV endonuclease incised cis-syn dimer-containing DNA in a dose-dependent manner and exhibited linear kinetics within that dose range. Enzyme activity was inhibited by the presence of NaCN or NaBH4 with NaBH4 additionally being able to trap a covalent enzyme-substrate product. These last findings confirm that the catalytic mechanism of M. luteus UV endonuclease, like those of other glycosylase/AP lyases, involves an imino intermediate.

Biological responses of two soybean cultivars exposed to enhanced UVB radiation

A UVB exposure and monitoring system has been established at the Oak Ridge National Laboratory's Global Climate Change Research Facility. The system consists of a power supply, and data acquisition and exposure equipment to accomplish controlled, elevated exposure of terrestrial plants to UVB. Plant biomass, selected compounds that absorb UV radiation, and DNA integrity/damage were measured for two soybean cultivars [ Glycine max (L.) Merr.] Forrest and Essex exposed to elevated UVB (32% above ambient) in this system. The biomass of each major plant organ was observed to be less in soybean cultivar Forrest upon exposure to enhanced UVB with the greatest response in seed pods and stems. In contrast, soybean cultivar Essex showed no biomass response to elevated UVB. Enhanced UVB caused significant ( P < 0.1) changes in concentrations of UV-absorbing compounds in both soybean cultivars. The Essex cultivar had an increase in UV-absorbing compounds, whereas a decline was observed for soybean Forrest. There was a decrease in the integrity of DNA, as measured by strand breaks, from both cultivars at 30 and 52 days to exposure. DNA pyrimidine dimers in isolated plant DNA were measured with Micrococcus luteus UV endonuclease. DNA from soybean Forrest exposed to UVB and sampled at 30 and 52 days of exposure had significantly greater ( P<0.05) pyrimidine dimer concentration (dimer frequency ≈ 1 dimer per 28,000 DNA bases) than either cultivar exposed to UV treatment for 1 day or Essex at days 30–52 (dimer frequencies < /1 per 120,000 bases of DNA). Decrease in DNA integrity and biomass production in Forrest under elevated UVB may be related to the inability to maintain high concentrations of UV-absorbing compounds in leaves. The tolerant cultivar Essex increased the concentratio of UV-absorbing compounds while maintaining biomass production and DNA integrity under elevated UVB.

UV endonuclease-mediated enhancement of UV survival in Micrococcus luteus: evidence revealed by deficiency in the Uvr homolog

Unlike its phage T4 counterpart (also known as endonuclease V), Micrococcus luteus UV endonuclease (pyrimidine dimer DNA glycosylase/apurinic-apyrimidinic endonuclease) has suffered from lack of genetic evidence to implicate it in the promotion of UV survival of the cell, i.e., mutants with its deficiency are no more UV-sensitive than the wild type. On the assumption that the contribution of UV endonuclease is obscured by the presence of a homolog of Escherichia coli UvrABC endonuclease, which has recently been identified in this bacterium, survival studies were carried out in its absence. With 254-nm UV irradiation, which generates not only pyrimidine dimers but also 6-4 photoproducts as lethal lesions, a double mutant defective in both UV endonuclease and the Uvr homolog was shown to be more sensitive than a single mutant defective only in the latter, with a dose reduction factor of approximately 2 at the survival level of 37%. Furthermore, molecular photosensitization, which produces only pyrimidine dimers, revealed an even greater difference in sensitivity, the dose reduction factor being about 3.4. These results indicate that the contribution to cell survival of UV endonuclease, an enzyme specific for pyrimidine dimers, is manifest if the backup by the Uvr homolog is absent.

Quantitation of ultraviolet radiation-induced cyclobutyl pyrimidine dimers in DNA by video and photographic densitometry

We have compared video and photographic methods for calculating the number of ultraviolet radiation (uv)-induced pyrimidine dimers in DNA from the bacteriophage T7 exposed to uv (0 to 800 J/m 2) from an FS40 sunlamp. DNA was incubated with a pyrimidine dimerspecific Micrococcus luteus uv endonuclease, subjected to alkaline agarose gel electrophoresis, neutralized, and stained with ethidium bromide, and the DNA fluorescence was recorded either with a video camera or on photographic film. The slopes of the dose-response curves for the number of uv-endonuclease-sensitive sites per 10 3 bases (pyrimidine dimers) was 1.2 (±0.1) × 10 −4 uv-endonuclease-sensitive sites per J/m 2 for the video analysis and 1.3 (±0.04) × 10 −4 uv-endonuclease-sensitive sites per J/m 2 for the photographic analysis. Results for pyrimidine dimer determination by either method were statistically comparable.

Bacteriophage-T4 and Micrococcus luteus UV endonucleases are not endonucleases but β-elimination and sometimes β δ-elimination catalysts

Bacteriophage-T4 UV endonuclease nicks the C(3')-O-P bond 3' to AP (apurinic or apyrimidinic) sites by a beta-elimination reaction. The breakage of this bond is sometimes followed by the nicking of the C(5')-O-P bond 5' to the AP site, leaving a 3'-phosphate end; delta-elimination is proposed as a mechanism to explain this second reaction. The AP site formed when this enzyme acts on a pyrimidine dimer in a polynucleotide chain undergoes the same nicking reactions. Micrococcus luteus UV endonuclease also nicks the C(3')-O-P bond 3' to AP sites by a beta-elimination reaction. No subsequent delta-elimination was observed, but this might be due to the presence of 2-mercaptoethanol in the enzyme preparation.

Superhelical stress restrained in plasmid DNA during repair synthesis initiated by the UvrA, B and C proteinsin vitro

Purified UvrA, UvrB, UvrC, UvrD, PolA and Lig proteins from Escherichia coli have been used to assess the effect of nucleotide excision repair on the conformation of native negatively supercoiled plasmid DNA in an in vitro test system. The analysis of labeled reaction products on specific gel systems suggests that the Uvr excinuclease has the ability to restrain the superhelical stress in the template DNA during the repair process. This feature, observed in the case of the Uvr system is not found if the repair reaction is initiated by T4 endonuclease V or Micrococcus luteus UV endonuclease.

Proximity of Repair Patches to Persistent Pyrimidine Dimers in DNA of Normal Human and Xeroderma Pigmentosum Cells

The proximity of repair patches to persistent pyrimidine dimers in normal human cells and xeroderma pigmentosum group C and D cells was analyzed by sequential digestion of repaired DNA with Micrococcus luteus UV-endonuclease and Escherichia coli DNA polymerase I. Although this enzymatic digestion removed one-third of the pyrimidine dimers, less than 3% of the label associated with repair patches and a similar amount of uniformly labeled DNA were removed. The repair patches therefore appear to be similarly distant from persistent dimers in all cell types, and, in particular, are not adjacent to unexcised dimers in xeroderma pigmentosum group D cells. A previous model that suggested that patches are inserted adjacent to dimers in xeroderma pigmentosum group D cells receives no support from these results.

Repair of UV-irradiated plasmid DNA in Saccharomyces cerevisiae Inability to complement mutational defects in excision repair by in vitro treatment with Micrococcus luteus UV endonuclease

Excision repair defects of Saccharomyces cerevisiae rad1-1, rad4-4, rad7-1 and rad14 mutants were examined. As previously found, transformation of such cells with UV-irradiated plasmid DNA is poor compared to wild-type yeast. Treatment of UV-irradiated YRp12 plasmid DNA with crude preparations of Micrococcus luteus UV endonuclease before introducing it into rad1-1 cells increased transformation efficiency to wild-type levels. This is consistent with earlier reports of rad1-1 mutants being defective in the incision step of excision repair. However, with purified UV endonuclease little or no rescue occured when the UV-irradiated plasmid was incised before transformation into rad1-1 or rad4-4 cells. Furthermore, the purified UV endonuclease reduced transformation of rad7-1 and rad14 mutants to levels seen in rad1-1 and rad4-4 cells. In contrast such treatment caused only a small decrease in the transforming ability of UV-irradiated DNA in wild-type cells. These results show that yeast can normally process pre-incised, UV-irradiated DNA and that this activity is absent in rad1-1, rad4-4, rad7-1 and rad14 mutants. Thus, in addition to their previously reported roles in incision, the RAD1, 4, 7 and 14 gene products are also required for repair to continue after the incision of DNA lesions.

Higher Pyrimidine Dimer Yields in Skin of Normal Humans with Higher UVB Sensitivity

We have measured UVB (280-320 nm)-induced DNA damage in skin of individuals with different sensitivities to UVB irradiation as measured by minimal erythema dose (MED). The DNA damage was susceptible to cleavage by Micrococcus luteus UV endonuclease, which recognizes pyrimidine dimers in DNA. An alkaline agarose gel electrophoresis method was used to quantitate the number of M. luteus UV endonuclease-sensitive sites in nonradioactive DNA from skin biopsies of 7 individuals irradiated with UVB (0-180 mJ X cm-2). The production of sites correlated well with MED (correlation coefficient = 0.78). The slope of the dose response curve for the most UVB-sensitive individual (MED = 24 mJ X cm-2) and for the least UVB-sensitive individual (MED = 146 mJ X cm-2) were 11.5 X 10(-4) and 2.6 X 10(-4) sites per 1000 bases per mJ X cm-2, respectively. The UVB-induced DNA damage was determined to be pyrimidine dimers by its susceptibility to cleavage by M. luteus UV endonuclease and its photoreactivability by Escherichia coli photoreactivating enzyme.

Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups

The UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles. In all complementation groups a restoration of the UV-induced UDS, in some cells to the repair-proficient human level, was observed. Another prokaryotic DNA-repair enzyme, T4 endonuclease V, restored the UV-induced UDS in a similar way after microinjection into XP cells. Since both enzymes specifically catalyse only the incision of UV-irradiated DNA, we conclude that this activity is impaired in cells of all 9 excision-deficient XP complemenation groups tested.

Repair of nonreplicating UV-irradiated DNA: cooperative dark repair by Escherichia coli uvr and phr functions.

The system previously used to study recombination of nonreplicating UV-irradiated phage lambda DNA was adapted to study UV repair. Irradiated phages infected undamaged homoimmune lysogens. Pyrimidine dimer content (by treatment with Micrococcus luteus UV endonuclease and alkaline sucrose sedimentation) and a biological activity endpoint (infectivity in transfection of uvrB recA recB spheroplasts) were followed. Unless room light was excluded during DNA extraction procedures, photoreactivation (Phr function) was significant. In uvr delta phr bacteria, repair, by both assays, was very low but not zero. Even when light was totally excluded, Phr function appeared to play a role in Uvr-mediated excision repair: both dimer removal and restoration of infectivity were two to five times as efficient in uvr+ phr+ bacteria as in uvr+ delta phr bacteria. Similarly, UV-irradiated phages plated with higher efficiencies on phr+ than delta phr bacteria even under totally dark conditions. In uvr phr+ repressed infections, removal of dimers from nonreplicating DNA did not increase infectivity as much as in uvr+ infections, suggesting a requirement for repair of nondimer photoproducts by the uvrABC system.

Kinetics of ultraviolet induced DNA excision repair in rat and human fibroblasts

To obtain more information on the well-documented low excision-repair capacity of rodent cells in comparison with human cells, we have studied this form of DNA repair in UV-irradiated human and rat skin fibroblasts. For this purpose, we have determined (i) unscheduled DNA synthesis (UDS), using autoradiography, (ii) the number and size of repaired sites with the bromodeoxyuridine (BrdU) photolysis assay and (iii) the removal of Micrococcus luteus UV-endonuclease susceptible sites (ESS). We found rat cells to be quite capable of performing DNA-repair synthesis, as demonstrated by both UDS and BrdU photolysis, whereas they almost completely lacked the capacity to remove pyrimidine dimers, as indicated by the persistence of ESS. This discrepancy will be discussed in terms of the types of mechanisms by which mammalian cells may recognize and remove UV-induced photoproducts.

Poly(adenosine diphosphoribose) synthesis in ultraviolet-irradiated xeroderma pigmentosum cells reconstituted with Micrococcus luteus UV endonuclease.

Synthesis of DNA and poly(adenosine diphosphoribose) [poly(ADPR)] was examined in permeabilized xeroderma pigmentosum lymphoblasts (XP3BE) before and after UV irradiation and in the presence and absence of Micrococcus luteus UV endonuclease. M. luteus UV endonuclease had no effect on the level of DNA or poly(ADPR) synthesis in control, unirradiated cells. UV irradiation caused a decrease in replicative DNA synthesis without any significant change in poly(ADPR) synthesis. In UV-irradiated cells treated with M. luteus UV endonuclease, DNA synthesis was restored to a level slightly greater than in the unirradiated control cells, and poly(ADPR) synthesis increased by 2- to 4-fold. Time--course studies showed that the UV endonuclease dependent poly(ADPR) synthesis preceded the endonuclease-dependent DNA synthesis. Inhibition of endonuclease-dependent poly(ADPR) synthesis with 3-aminobenzamide, 5-methylnicotinamide, or theophylline produced a partial inhibition of the endonuclease-dependent DNA synthesis. Conversely, inhibition of the endonuclease-dependent DNA synthesis with dideoxythymidine triphosphate, phosphonoacetic acid, or aphidicolin had no effect on the endonuclease-dependent poly(ADPR) synthesis. These studies show that stimulation of poly(ADPR) synthesis in UV-irradiated cells occurs subsequent to the DNA strand breaks created by the specific action of the UV endonuclease on UV-irradiated DNA. The effect of the inhibitors of poly(ADPR) synthesis in UV-irradiated cells indicates that the endonuclease-stimulated DNA synthesis is dependent in part on the prior synthesis of poly(ADPR).

Strand cleavage at psoralen adducts and pyrimidine dimers in DNA caused by interaction between semi-purified uvr+ gene products from Escherichia coli

Partially purified extracts of Escherichia coli containing either uvrA + or a mixture of uvrB + and uvrC + gene products were tested for an endonuclease activity on DNA treated with 8-methoxypsoralen plus 360-nm light. Neither of these fractions was active alone. The combined fractions, however, caused extensive strand cleavage of the psoralen-treated DNA. The endonuclease activity was dependent upon addition of ATP and Mg 2+ to the reaction mixtures, and hence appeared similar to the UV-endonuclease activity previously shown to be reconstituted from the same fractions. It is concluded that the uvr + gene products in these fractions interact to cause breakage of both psoralen-treated and UV-irradiated DNA. An examination of the dose-dependence relationship of the break formation in psoralen-treated DNA revealed that the enzyme acts upon psoralen mono-adducts. By varying the experimental conditions to increase the ratio of interstrand cross-links to mono-adducts it was found that the enzyme also acts upon cross-links, but with lower efficiency than for mono-adducts. Further studies of break formation in UV-irradiated DNA showed that elimination of pyrimidine dimers by treatment with photoreactivating enzyme in the light resulted in a loss of endonuclease-sensitive sites. This shows directly that pyrimidine dimers are the lesions recognized by the complemented uvr + gene products in UV-irradiated DNA. For comparison, another endonuclease acting at pyrimidine dimers in DNA, the Micrococcus luteus UV-endonuclease, was also tested with psoralen-treated DNA, but no activity was observed. This and other data indicate that the repair endonuclease encoded by the uvr + genes in E. coli is basically different from the other dimer-specific endonucleases previously characterized.

Repair of 2 um Plasmid DNA in Saccharomyces cerevisiae

We have developed a system for assaying pyrimidine dimers in the 2 ⇐m DNA plasmid of Saccharomyces cerevisiae, using Micrococcus luteus UV endonuclease to nick dimer-containing plasmid molecules and measuring percentages of nicked and covalently closed circles on agarose gels. UV-irradiation induced dimers in plasmid DNA, in vivo, at the same rate as in chromosomal DNA. After a dose of 20 Joules·m(-2), approximately 86% of plasmid molecules had. at least one dimer. After 3 h incubation under normal growth conditions only 4% still retained dimers in a wild-type strain. In a rad1 (excision-defective) mutant 81% of plasmid molecules still had dimers after 3 h, suggesting that excision repair operates to remove dimers from plasmid DNA in wild-type yeast. Dimers can be removed from 2 ,um DNA in a rad1 mutant by photoreactivation.

Substrate specificity of Micrococcus luteus UV endonuclease and its overlap with DNA photolyase activity.

The action of an endonuclease from Micrococcus luteus that operates on UV damage in DNA overlaps with that of DNA photolyase from yeast: homo- and heterocyclobutane dipyrimidines in DNA are substrates 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 photolyase-cyclobutane dipyrimidine complex can prevent nicking by the endonuclease.