Photoreactivation of template activity of UV—irradiated DNA in an RNA—polymerase system. A rapid assay for photoreactivating enzyme

Piessens, J.P.; Eker, A.P.M.

doi:10.1016/0014-5793(75)80471-6

Cited by 15 publications

(6 citation statements)

References 19 publications

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“…-2 10 withdrawn from the cuvette were immediately frozen in CO,/acetone and stored at -20°C until the transformation assay was performed (for experimental details see Piessens and Eker, 1975;Eker, 1980). The incident light flux was adapted and corrected for absorption of the sample according to Morowitz (1950) which is particularly important in the UV region.…”

mentioning

confidence: 99%

PHOTOREACTIVATING ENZYME FROM Streptomyces griseus—VI. ACTION SPECTRUM AND KINETICS OF PHOTOREACTIVATION

Eker

Hessels

Dekker

1986

Photochem & Photobiology

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Abstract— A high resolution action spectrum for photoreactivation was determined using purified photoreactivating enzyme from Streptomyces griseus. Conversion of pyrimidine dimers in UV‐irradiated DNA, the substrate for photoreactivating enzyme, was measured with a Haemophilus influenzae transformation assay. A high similarity was found between action spectrum (max. at 445 nm) and the long wavelength absorption band (max. at 443 nm)of photoreactivating enzyme. In addition to the400–470 nm region considerable photoreactivation was found with wavelengths between 280 and 320 nm. No evidence was obtained for the presence of nonenzymatic photoreactivation. Comparison of in vitro and in vivo action spectra revealed that the sharp peak at 313 nm found in vivo is probably the result of counteracting photoreactivation and inactivation effects. Comparison of the action spectrum with the absorption spectrum of 8‐hydroxy‐10‐methyl‐5‐deazaisoalloxazine in an aprotic dipolar solvent (which serves as a model for the 8‐hydroxy‐5‐deazaflavin chromophore in photoreactivating enzyme) indicates the possible presence of other chromophore(s) involved in the photorepair process. From kinetic measurements and flash experiments values were obtained for the rate constants of the photoreactivation reaction. The quantum yield of photoreactivation was estimated to be approximately 1.

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confidence: 99%

PHOTOREACTIVATING ENZYME FROM Streptomyces griseus—VI. ACTION SPECTRUM AND KINETICS OF PHOTOREACTIVATION

Eker

Hessels

Dekker

1986

Photochem & Photobiology

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“…Assay of photoreactivating activity. Activities of PRE were measured in vitro with the Haemophilus infruenrae transformation assay (Piessens and Eker, 1975;Eker, 1980). Enzyme activity is expressed as DF(N, -ND)/ND in which DF is the enzyme dilution factor and N D and NL are the number of transformants obtained with dark incubated and photoreactivated samples respectively.…”

Section: Ultraviolet-inactivation H Cutirubrum Cells Were Grownmentioning

confidence: 99%

PHOTOREACTIVATION IN THE EXTREME HALOPHILIC ARCHAEBACTERIUM Halobacterium cutirubrum

Eker¹,

Formenoy²,

Wit³

1991

Photochem Photobiol

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Photoreactivation in the extreme halophilic archaebacterium Halobacterium cutirubrum was studied both in vivo and in v i m . Cells irradiated with ultraviolet (UV)-fluences up to 350 JimZ could be completely photoreactivated, indicating very efficient repair of pyrimidine dimers in UV-irradiated DNA. Dark repair is apparently absent in Halobacterium since liquid holding under non-growth conditions did not influence the survival of UV-irradiated cells, while cells remained completely photoreactivable with no change in the kinetics of photoreactivation. Experiments with Halobacterium isolates of different carotenoid content indicated that carotenoids do not influence either UV-inactivation or photoreactivation. Small differences in the rates of UV-inactivation and photoreactivation could be assigned to the occurrence of gas vesicles. Flash experiments and the temperature dependence of photoreactivation indicated an enzymatical reaction. This was confirmed by in vitro experiments with partially purified photoreactivating enzyme. The in vivo action spectrum of photoreactivation showed a main band in the 400470 nm region with a maximum at 440 nm. Comparison with action spectra of other microorganisms classified the Halobacterium enzyme as a 8-hydroxy-5-deazaflavin type photoreactivating enzyme.

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“…Purification of yeast PRE has been described previously (Zwetsloot et al, 1985). The microinjected enzyme is highly active in the in vitro Haemophilus influenzae transformation assay (Rupert et al, 1958;Zwetsloot et al, 1985; for experimental details see Piessens and Eker, 1975).…”

Section: Purification Of Prementioning

confidence: 99%

Unscheduled DNA synthesis in xeroderman pigmentosum cells after microinjection of yeast photoreactivity enzyme

Zwetsloot

Hoeymakers

Vermeulen

et al. 1986

Mutation Research/DNA Repair Reports

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Photoreactivating enzyme (PRE) from yeast causes a light-dependent reduction of UV-induced unscheduled DNA synthesis (UDS) when injected into the cytoplasm of repair-proficient human fibroblasts (Zwetsloot et al., 1985). This result indicates that the exogenous PRE monomerizes UV-induced dimers in these cells competing with the endogenous excision repair. In this paper we present the results of the injection of yeast PRE on (residual) UDS in fibroblasts from different excision-deficient XP-strains representing complementation groups A, C, D, E, F, H and I (all displaying more than 10% of the UDS of wild-type cells) and in fibroblasts from two excision-proficient XP-variant strains. In fibroblasts belonging to complementation groups C, F and I and in fibroblasts from the XP-variant strains UDS was significantly reduced, indicating that pyrimidine dimers in these cells are accessible to and can be monomerized by the injected yeast PRE. The UDS reduction in the XP-variant strains is comparable with the effect in wild-type cells. In cells from complementation groups C, F and I the reduction is less than in wild-type and XP-variant cells. Fibroblasts belonging to groups A, D, E and H did not show any reduction in UDS level after PRE injection and illumination with photoreactivating light. These results give evidence that the genetic repair defect in some XP-strains is probably due to an altered accessibility of the UV-damaged sites.

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Photoreactivation of template activity of UV—irradiated DNA in an RNA—polymerase system. A rapid assay for photoreactivating enzyme

Cited by 15 publications

References 19 publications

PHOTOREACTIVATING ENZYME FROM Streptomyces griseus—VI. ACTION SPECTRUM AND KINETICS OF PHOTOREACTIVATION

PHOTOREACTIVATING ENZYME FROM Streptomyces griseus—VI. ACTION SPECTRUM AND KINETICS OF PHOTOREACTIVATION

PHOTOREACTIVATION IN THE EXTREME HALOPHILIC ARCHAEBACTERIUM Halobacterium cutirubrum

Unscheduled DNA synthesis in xeroderman pigmentosum cells after microinjection of yeast photoreactivity enzyme

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