GENETICALLY MODIFIED BACTERIA PRODUCING THREE DNA REPAIR ENZYMES AND METHOD FOR THE EVALUATION OF DNA REPAIR ACTIVITY
20220042024 · 2022-02-10
Inventors
Cpc classification
C12Y401/99003
CHEMISTRY; METALLURGY
C12Q1/683
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
G01N2333/988
PHYSICS
International classification
C12N15/70
CHEMISTRY; METALLURGY
Abstract
Three recombinant E. coli strains produce the enzymes CPD-photolyase, 6,4-bifunctional photolyase and 6,4-photolyase, from bacterial Antarctic isolates of the genus Hymenobacter the first one and Sphingomonas the others. It is also disclosed a process of production and purification of the recombinant enzymes with high performance, high degree of purity and high catalytic repair activity, having applications in, but it is not limited to, cosmetics and pharmaceutical industry. A fast, cheap and qualitative method is provided for the determination of the CPD photolyase activity.
Claims
1. A method for the production of at least one recombinant photolyase selected from CPD-photolyases, 6,4-bifunctional photolyases and 6,4-photolyases that comprises the transformation of Escherichia coli bacteria with the host vector pET28a (+) comprising one optimized gene sequence selected from bacterial Antarctic isolates of the genus Hymenobacter for the CPD photolyase and Sphingomonas for the 6,4-photolyase and the 6,4-bifunctional photolyase, culturing for their expression and purification.
2. The method of claim 1, wherein the at least one recombinant photolyase is selected from CPD-photolyases of SEQ ID No2, 6,4-bifunctional photolyase of SEQ ID No 4, and 6,4-photolyase of SEQ ID No6 that comprises the transformation of Escherichia coli bacteria with the host vector pET28a (+) comprising the optimized gene sequence selected of SEQ ID No1, SEQ ID No3, and SEQ ID No5 culturing for their expression and purifying them, wherein said optimized gene sequence comes from the Hymenobacter sp. UV11 for CPD photolyases and Sphingomonas sp. UV9 for 6,4-bifunctional photolyase and 6,4-photolyase, respectively.
3. A recombinant Escherichia coli bacteria transformed in the method of claim 1, comprising the optimized sequence selected of SEQ ID No1, SEQ ID NO 3 and SEQ ID No 5.
4. The recombinant Escherichia coli bacterium of claim 3, wherein the recombinant Escherichia coli bacterium is designated as PhotoUV11.
5. The recombinant Escherichia coli bacterium of claim 3, wherein the recombinant Escherichia coli bacterium is designated as PhotoUV9.
6. The recombinant Escherichia coli bacterium of claim 3, wherein the recombinant Escherichia coli bacterium is designated as PhotoSphingo97.
7. The recombinant Escherichia coli bacteria of claim 4, wherein the recombinant Escherichia coli bacterium produce CPD-photolyases with high performance and high degree of purity, showing high CPD repair activity, for pharmaceutical and cosmetic uses.
8. The recombinant Escherichia coli bacteria of claim 5, wherein the recombinant Escherichia coli bacterium produce 6,4-bifunctional photolyases with high performance and high degree of purity, showing both CPD and 6,4 photoproducts DNA lesions repair activities.
9. The recombinant Escherichia coli bacteria of claim 6, wherein the recombinant Escherichia coli bacterium produce 6,4-photolyases with high performance and high degree of purity, showing 6,4 photoproducts DNA lesions repair activity.
10. An isolated nucleic acid molecule encoding photolyases consisting of the amino acid sequence of SEQ ID No2, SEQ ID No4 or SEQ ID No6.
11. The isolated nucleic acid molecule of claim 10 comprising the sequence of SEQ ID No1.
12. The isolated nucleic acid molecule of claim 10 comprising the sequence of SEQ ID No3.
13. The isolated nucleic acid molecule of claim 10 comprising the sequence of SEQ ID No5.
14. A recombinant CPD-photolyase produced from the recombinant Escherichia coli bacterium of claim 7, comprising an amino acid sequence of SEQ ID No2, with high degree of purity and showing high CPD-product repair activity.
15. A recombinant 6,4-bifunctional photolyase produced from the recombinant Escherichia coli bacterium of claim 8, comprising an amino acid sequence of SEQ ID No 4 showing high degree of purity and high CPD and 6,4-photoproducts DNA lesions repair activities.
16. A recombinant 6,4-photolyase produced from the recombinant Escherichia coli bacterium of claim 9, comprising an amino acid sequence of SEQ ID No 6 showing high degree of purity and high 6,4-photoproducts DNA lesions repair activity.
17. A method for the determination of qualitative CPD-photolyase activity consisting in: UVC-irradiating the plasmid puc18-2 with 1000 J m.sup.−2 using a Spectroline lamp; evaluating the full radiation by MseI digestion and 1% agarose-gel electrophoresis at room temperature, for 1 h at 60V, using TAE as running buffer; visualizing DNA-restriction fragments by staining with ethidium bromide; and determining the DNA-damage by the absence of the 780 kbp DNA fragment corresponding to the miniTn5 cassette when the irradiated plasmid pUC18-2 is 25 previously incubated with CPD-photolyases.
Description
BRIEF DESCRIPTION OF DRAWINGS AND FIGURES
[0022] Table 1—DNA repair assay (Comet assay) using Chinese Hamster Ovary (CHO) cells (experiments carried on with the CPD-photolyase enzyme)
[0023] Table 2—DNA repair assay (Comet assay) using immortalized non-tumorigenic human epidermal. (HaCat) cells experiments carried on with the CPD-photolyase enzyme)
[0024] Table 3—CPD-repair activity by immunoassay using the CPD-photolyase enzyme from Hymenobacter sp. UV11)
[0025] Table 4—6,4-photoproduct-repair activity by immunoassay (using the 6,4-bifunctional photolyase, enzyme from Sphingomonas sp. UV9)
[0026] Table 5—CPD-repair activity by immunoassay (using the 6,4-bifunctional photolyase enzyme, from Sphingomonas sp. UV9)
[0027] Table 6—6,4-photoproduct-repair activity by immunoassay using the 6,4-photolyase, enzyme from Sphingomonas sp. UV9).
[0028]
[0029]
[0030]
[0031]
[0032]
[0033]
[0034] DNA repair assays (Comet assay) using the Chinese Hamster ovary (CHO) cell line.
[0035] Asterisks indicate significant difference among treatments (P<0.05).
[0036]
[0037]
[0038]
[0039]
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention provides three recombinant E. coli strains for the production of CPD or 6,4, or bifunctional-pholotyases with the following characteristics:
[0041] (A) The recombinant production of a CPD-photolyase from a bacterial Antarctic isolate of the genus Hymenobacter.
[0042] (B) The recombinant production of a 6,4-bifunctional photolyase from a bacterial Antarctic isolate of the genus Sphingomonas.
[0043] (C) The recombinant production of a 6,4-photolyase from a bacterial Antarctic isolate of the genus Sphingomonas.
[0044] In addition, the invention provides the method of production and purification with high yield of a CPD-photolyase, a 6,4-bifunctional photolyase and a 6,4-photolyase, with DNA repair activity, and a method for the determination of CPD-photolyase activity.
[0045] The invention also provides CPD-photolyase, 6,4-bifunctional photolyase and 6,4-photolyase enzymes from bacterial Antarctic isolates of the genus Hymenobacter and Sphingomonas, respectively, all three enzymes with high purity and the compositions containing them.
[0046] Method for the Production of E. coli Recombinant Strains
[0047] (A) The CPD-photolyase gene sequence was obtained from the draft cenome of Hymenobacter sp. UV11, optimized for their expression in E. coli BL21 (DE3), and synthesized in the host-vector pET28a(+) (
[0048] (B) The 6,4-bifunctional photolyase gene sequence was obtained from the draft genome of Sphingomonas sp. UV9, optimized for their expression in E. coli BL21 (DE3), and synthesized in the host-vector pET28a(+) (
[0049] (C) The 6,4-photolyase gene sequence was obtained from the draft genome of Sphingomonas sp. UV9, optimized for their expression in E. coli BL21 (Arctic), and synthesized in the host-vector pET28a(+) (
[0050] The resulting strains of Escherichia coli were called PhotoUV11, deposited at CECT (Number CECT 9643), PhotoUV9, deposited at CECT (Number CECT 9642), and PhotoSphingo97, deposited at CChRGM (accession number at the form R0134), respectively.
[0051] All three recombinant enzymes, CPD-photolyase, 6,4-bifunctional photolyase and 6,4-photolyase were produced in the auto-inductor Zym-5052 medium and purified by immobilized Metal Affinity Chromatography (IMAC) as active enzymes as shown by Comet Assay (using CHO and HaCat cell lines) (Tables 1 and 2; and Immunochemistry Detection of photoproducts (Tables 3, 4 and 5).
[0052] We have also found and demonstrated that the recombinant 6,4-bifunctional photolyase produced by the process here described shows both CPD- and 6,4-photoproduct-repair activities indicating the potential use of this enzyme in the development of new products with pharmaceutical and cosmetic applications.
[0053] This invention also describes a simple and fast method to discriminate between active and non-active C2D-photolyases.
[0054] The method for the determination of CPD-photolyase activity was developed using a plasmid (pUC18-2) as DNA substrate and can be performed using basic laboratory equipment. This plasmid holds a kanamycin resistance cassette (miniTn5) flanked by TTAA sites (sites of CPD formation). The plasmid (UVC-irradiated and non-irradiated samples) was digested with the MseI restriction enzyme (that recognized TTAA sequences) and subjected to an agarose-gel electrophoresis. When properly UVC irradiated, the TTAA sequences are blocked by the formation of cyclobutane pyrimidine dimers and the MseI enzyme cannot recognize the restriction site and the miniTn5 cassette cannot be released. CPD-activity this was determined by the comparison of the electrophoretic mobility, or restriction pattern, of non-irradiated and UVC-irradiated pUC18-2 samples after the incubation with the CPD-photolyase and further digestion with MseI. Only if the CPDs are repaired by the CPD-photolyase, the miniTn5 cassette was observed as a discrete DNA band in the agarose-gel.
[0055] Description of the E. coli Developed Strains:
[0056] The (A) CPD-photolyase Class I gen from Hymenobacter sp. UV11 (GenBank Accession Number KX118295), (B) 6,4-bifunctional photolyase gen from Sphingomonas sp. UV9 (GenBank Accession Number KX1182981 and (C) 6,4-photolyase gen from Sphingomonas sp. UV9 (GenBank Accession Number KX118297) were modified, synthesized fused in the plasmid vector pET28a(+) (encoding an N-terminal 6-His tag) and transformed into Escherichia coli BL21 (DE3) or Escherichia coli BL21 (Arctic; cells, using the calcium chloride protocol (Sambrook J., Fritsch E. F., Maniatis T. 1989. Molecular Cloning: A Laboratory Manual, second ed., vol. 3. OSH Laboratory Press, Cold Spring Harbor, N.Y.).
[0057] Modifications made to the gene sequences include the change in 20% of the nucleotides. The codon usage was modified by updating the Codon Adaptation Index (CAI) from 0.51 to 0.96 for CPD-photolyase and 6,4-photolyase and from 0.52 to 0.97 for the 6,4-bifunctional photolyase. The GC content was reduced from 61.29% to 56.60%, 65.54% to 59.22% and from 69.06% to 64.27%, respectively.
[0058] The constructs (the recombinant plasmid) were synthesized by GenScript (https:www.genscript.com/; USA). The codon optimization for the gene expression in E. coli were performed for the CPD-photolyase Class I gene from Hymenobacter sp. UV11 (Gen Bank Accession Number KX118295), for the 6,4-bifunctional photolyase gene from Sphingomonas sp. UV9 (GenBank Accession Number KX118296) and for the 6,4-photolyase gene from Sphingomonas sp. UV9 (GenBank Accession Number KX118297). The optimized genes were fused separately into the plasmid vector pET23a(+) in each case; the expression vectors encode for the production of the photolyases with an N-terminal C-His tag.
[0059] For plasmid maintenance, the construct in each case was transformed to E. coli DH5α chemo competent cells and inoculated on Luria-Bertoni (LB) plate containing 50 μg/ml of Kanamycin. Plasmids were transformed using the calcium chloride protocol as described by Sambrook et al. 0.1989 (Molecular Cloning: A Laboratory Manual, second ed., vol. 3. CSH Laboratory Press, Cold Spring Harbor, N.Y. Cells were stored in 15% glycerol at −80° C.).
[0060] For ax situ microbial repository purpose in an International Depositary Authority (IDA) under the 1977 Budapest treaty on the International Recognition of the deposit of Microorganisms, the recombinant strains were deposited as Escherichia coli BL21(DE5) PhotoUV11(A) and PhotoUV9 (B), respectively, in Colección Española Cultivos Tipo (CECT) (parc Cientific Universitat de Valencia, Spain); and Escherichia coli BL21 (Arctic) PhotoSphingo97 (C) in Colección Chilena de Fecursos Genéticos Microbianos (CChRGM) (Avenida Vicente Mendez No 515, Chillan, Chile).
[0061] For protein production, precultures were obtained by growth on Luria Broth at 37° C. Cells were transferred to the auto-inductor Zym-5052 medium (Studier, F. W. 2005. Protein production by auto-induction in high-density shaking cultures. Protein expression and purification. 41: 207-234), containing 50 μg/ml Kanamycin, and growth at 14° C. and 200 rpm, for 48 h. Cells were harvested, washed twice with PBS (1.37 M calcium chloride, 27 mM potassium chloride, 100 mM sodium dibasic phosphate, 18 mM potassium monobasic phosphate), lysed by sonication (40% Amplitude, at a relative power output of 10) using 50 mM phosphate buffer (supplemented with 50 mM NaCl, at pH and centrifuged twice, first at 7000 g for 10 min at 4° C., and then at 16000 g for 30 min at 4° C.
[0062] Photolyases were purified by binding the soluble fraction of proteins at a Ni-NTA affinity resin in binding buffer 150 mM phosphate buffer containing 50 mM NaCl for CPD-photolyase and 6,4-bifunctional. photolyase, and 300 mM NaCl for 6,4-photolyase, supplemented with 50 mM Imidazole for CPD-photolyase and 6,4-bifunctional photolyase, and 10 mM imidazole for 6,4-photolyase) for 1. h at 4° C., washed with binding buffer, eluted with binding buffer containing 250 mM Imidazole and further desalted using Desalting Columns contain Sephadex G-25 resin. All fractions were controlled by SDS-PAGE (
[0063] The yield of the process was 42 mg of a 50 kDa protein in 6 ml corresponding to the recombinant CPD-photolyase from Hymenobacter sp. UV11 (A) (Protein sequence SEQ ID No 2), 70 mg of a 54 kDa protein in 6 ml corresponding to the 6,4-bifunctional photolyase from Sphingomonas sp. UV9 (B) (Protein sequence SEQ ID 4) and 40 mg of a 59 kDa protein in 6 ml corresponding to the 6,4-photolyase from Sphingomonas sp. UV9 (C) (Protein sequence SEQ ID 6), each produced from 200 ml culture.
[0064] The identity of the recombinant protein was verified by mass spectrometry MALDI/TOFTOF analysis and searching in the NCBI database with peptide m/z values using the MASCOT software.
[0065] The activity of the recombinant CPD-photolyase from Hymenobacter sp. UV11 was demonstrated in in vitro experiments using comet and immune assays. For comet assay CHO and HaCat cell lines were used (Tables 1 and 2). HaCat or CHO cell samples were washed twice with cold PBS, irradiated with a dose of 4 J m.sup.−2 UVC radiation (254 nm) using a Spectroline lamp (model ENF-260C/FE), washed again, scraped using a rubber policeman and transferred to eppendorf tubes with 1 ml of PBS. Twenty μI of cell suspension (2×10.sup.−6 cells) were gently mixed with 80 μI of 1% Low melting-point agarose (LMA), and directly applied to a 1.5% agarose precoated slide. Slides were covered with a coverslip and placed at 4° C. for 5 min. Then, coverslips were removed and slides were submersed in the lysis solution (2.5 M NaCI, 100 mM Na.sub.2EDTA, 1 mM Tris buffer at pH 10, 1% Triton X-100) and incubated at 4° C. for 2 h. Slides were washed twice with buffer NET (100 mM NaCI, 10 mM Tris-HCI, 10 mM EDTA; pH 8.0) for 5 min at room temperature. When indicated (treatments with photolyase), 50 μI of recombinant CPD-photolyase at different concentrations (suspended in NET buffer) was applied to the top of gels, irradiated with a UVA lamp (ULTRA-VITALUX OSRAM 300W) for 10 min in a humidity chamber (photoreparation), and then washed twice in buffer: NET. Ail slides (treatments and controls) were loaded with 50 uI of T4 endonuclease V (in buffer NET) (the enzyme cleaves the glycosyl bond of the 5′end of the pyrimidine dimer; unrepaired CPDs are revealed by this enzyme), covered with a coverslip and incubated in a humidified chamber for 30 min at 37° C. DNA fragments were resolved by alkaline-electrophoresis, using the following running buffer: 1 mM Na.sub.2EDTA, 0.3 NaOH, at pH 13. DNA was unwinded for 15 min and then, the electrophoresis was performed at 0.7 V/cm, 300 mA for 20 min, in a cold unit at 4° C. The slides were removed and washed in neutralization buffer (0.4 M Tris-HCI; pH 7.5) for 5 min at room temperature, and finally stained with 50 μL ethidium bromide (10 mg/ml). Slides were analyzed using the Computer Program Comet Imager (MetaSystems). At least 50 nuclei per slide were measured (three slides per treatment or control). Three independent biological replicas were performed. CHO (Table 1,
[0066] The recombinant CPD-photolyase also showed activity by immunochemistry assay. Immunochemistry of irradiated Calf thymus DNA was performed to quantify CPDs using the High Sensitivity CPD/Cyclobutane Pyrimidine Dimer ELISA kit of CosmoBio (NMMA-K001), with modifications as follows. UVC-irradiated calf thymus DNA. (50 μL of J, 4 μg/ml DNA irradiated with 10 J) was denatured (20 min at 100° C., followed by chilling on ice for 15 min), applied to microtiter wells pre-coated with protamine sulfate and then treated with 150 μL of the recombinant photolyase at different concentrations during 20 min under blue light at room temperature. Experiments were also carried out in darkness conditions. Then, the photolyase was removed by washing with washing buffer, treated with specific monoclonal anti-CPLD antibody clone TDM-2 and washed again. The remaining TDM-2 antibody in each well was then measured by sequential treatment with a secondary biotinylated antibody, streptavidin-peroxidase, and the substrate 3,3′,5,5′-tetramethylbenzidine (TMB). The final product develops a color, which was measured at 450 nm. Reactive compounds, including the irradiated calf thymus DNA, antibodies and buffers, were used following manufacturer's instructions (CosmoBio). Results are shown in Table 3 and
[0067] The Immunochemical. assay experiments, using commercial antibodies that recognize CPDs, also confirmed that the recombinant CPD-photolyase was purified as an active DNA repairing enzyme. In these experiments, calf-thymus DNA (single stranded DNA) was used as substrate and the recombinant photolyase reduced by 100, the presence of CPDs (UVC-irradiated and non-photolyase treated DNA was considered at an initial damage of 100%). We detected repair activity even by using 1 μg/ml recombinant CPD-photolyase (a 5000-fold dilution of the purified enzyme). When experiments were carried out on darkness, or when the recombinant enzyme was denatured by heat (boiling for 10 min and fast cooling on ice), the repair activity was not detected, discarding false positives or a method artifact. These results support that the recombinant CPD-photolyase from Hymenobacter sp. UV11 has been produced as active enzyme, and it needs light for activity. 6,4-photoproducts antibodies were also tested in immunochemical assays, and results suggested that the recombinant CPD-photolyase might not repair this Kind of DNA lesion.
[0068] The activity of the recombinant; 6,4-bifunctional photolyase from Sphingomonas sp. UV9 was demonstrated in in vitro experiments by using the immunoassay. Both CPD- and 6,4-antibodies were used as described above, reparation the High sensitivity ELISA kit for measuring UV-induced DNA damage from CosmoBio CSR-NM-MA-K004) was used for detecting 6,4-photoproducts. The information (Table 4 and 5) supports that the recombinant 6,4-bifunctional photolyase from Sphingomonas sp. UV9 has both CPD- and 6,4-photoproduct repair activities.
[0069] The activity of the recombinant 6,4-photolyase from Sphingomonas sp. UV9 was demonstrated in in vitro experiments by using the immunoassay. The High sensitivity ELISA kit for measuring UV-induced DNA damage from CosmoBio (CSR-NM-MA-K004), using the 6,4-antibodies, was used. The information (Table 6 and
[0070] Finally, the recombinant photolyases were stored in PBS buffer supplemented with 50% glycerol at −80° C. and remained 100% active at least after one-year storage in this conditions.
EXAMPLES
Example 1—DNA Repair Assay (Comet Assay) Using Chinese Hamster Ovary Cells
[0071] Olive tail moments and statistics. Experiments were performed using the Chinese Hamster Ovary (CHO) cell line.
[0072] It was observed that UVC-irradiated CHO cells that were treated with the recombinant photolyase in the presence of blue light had a significantly lower value of olive tail moment rather than non-photolyase treated cells, suggesting that under the assay conditions the recombinant CPD-photolyase from Hymenobacter sp. UV11 reduced the UVC-DNA damage of CHO cells. This photolyase almost completely repaired the DNA damage caused by UVC radiation.
[0073] These results were supported by the statistic test (multiple comparison ANOVA assay), where 50 comets per treatment were counted in duplicate.
TABLE-US-00001 TABLE 1 Standard Mean error for the Test details Mean 1 Mean 2 Difference difference Significance Summary C.sub.1− vs. 1.321 1.132 0.189 0.6291 No ns C.sub.2− C.sub.1− vs. 1.321 4.838 −3.517 0.7705 Yes **** C.sub.3− C.sub.1− vs. 1.321 37.27 −35.95 0.7705 Yes **** C+ C.sub.1− vs. 1.321 4.869 −3.548 0.7121 Yes **** Photo_1 C.sub.2− vs. 1.132 4.838 −3.706 0.7705 Yes **** C.sub.3− C.sub.2− vs. 1.132 37.27 −36.14 0.7705 Yes **** C+ C2− vs. 1.132 4.869 −3.737 0.7121 Yes **** Photo_1 C.sub.3− vs. 4.838 37.27 −32.44 0.8897 Yes **** C+ C.sub.3− vs. 4.838 4.869 −0.031 0.8396 No ns Photo_1 C+ vs. 37.27 4.869 32.4 0.8396 Yes **** Photo_1 Abbreviations are as follows: C1−, non-irradiated cells (non-damaged DNA); C2−, non-irradiated cells treated with endonuclease T4 V (basal DNA damage under normal conditions); C3−, UVC-irradiated cells (damaged DNA); C4−, non-irradiated cells treated with the recombinant photolyase (potential damage 5 produced by the recombinant photolyase); C+, UVC-irradiated cells treated with endonuclease T4 V (total damage of DNA); Photo_1, Photo_0.1 and Photo_0.02 are cells treated with 50 μI of 1, 0.1 and 0.02 mg/ml recombinant CPD photolyase.
Example 2—DNA Repair Assay (Comet Assay) Using Immortalized Non-Tumorigenic Human Epidermal HaCat Cells
[0074] Olive tail moments and statistics experiments were performed using the HaCat cell line. Results showed reduced values of olive tail moments, similar to those values found for the cells that were not exposed to UVC radiation suggesting that under the assayed conditions the recombinant CPD-photolyase from Hymenobacter sp. UV11 reduced the JVC-DNA damage of HaCat cells almost completely.
[0075] These results wee supported by the statistic test (multiple comparison ANOVA assay), where 50 comets per treatment were counted in duplicate.
TABLE-US-00002 TABLE 2 Standard Mean error for the Test details Mean 1 Mean 2 Difference difference Significance Summary C+ vs. C− 2.348 0.1655 2.182 0.03399 Yes **** C+ vs. 2.348 0.26 2.088 0.03399 Yes **** Photo_2 + BL C+ vs. 2.348 0.2475 2.1 0.03399 Yes **** Photo_1.5 + BL C+ vs. 2.348 0.266 2.082 0.03399 Yes **** Photo_1 + BL C+ vs. 2.348 2.351 −0.003 0.03399 No ns Photo_1.5 Heat C+ vs. 2.348 2.137 0.151 0.03399 Yes * Photo_1.5 C+ vs. 2.348 2.251 0.097 0.03399 No ns Photo_1 C− vs. 0.1655 0.26 −0.0945 0.03399 No ns Photo_2 + BL C− vs. 0.1655 0.2475 −0.082 0.03355 No ns Photo_1.5 + BL C− vs. 0.1655 0.266 −0.1005 0.03399 No ns Photo_1 + BL C− vs. 0.1655 2.351 −2.185 0.03399 Yes **** Photo_1.5 Heat C− vs. 0.1655 2.197 −2.031 0.03399 Yes **** Photo_1.5 C− vs. 0.1655 2.251 −2.085 0.03399 Yes **** Photo_1 Photo_2 + BL 0.26 0.2475 0.0125 0.03399 No ns vs. Photo_1.5 + BL Photo_2 + BL 0.26 0.266 −0.006 0.03399 No ns vs. Photo_1.5 + BL Photo_2 + BL 0.26 2.351 −2.091 0.03399 Yes **** vs. Photo_1.5 Heat Photo_2 + BL 0.26 2.197 −1.937 0.03399 Yes **** vs. Photo_1.5 Photo_2 + BL 0.26 2.251 −1.991 0.03399 Yes **** vs. Photo_1 Photo_1.5 + BL 0.2475 0.266 −0.0185 0.03399 No ns vs. Photo_1 + BL Photo_1.5 + BL 0.2475 2.351 −2.103 0.03399 Yes **** vs. Photo_1.5 Heat Photo_1.5 + BL 0.2475 2.197 −1.949 0.03399 Yes **** vs. Photo_1.5 Abbreviations are as follows: C1−, non-irradiated cells (non-damaged DNA); C2−, non-irradiated cells treated with endonuclease T4 V (basal DNA damage under normal conditions); C3−, UVC-irradiated cells (damaged DNA); C4−, non-irradiated cells treated with the recombinant photolyase (potential damage 5 produced by the recombinant photolyase); C+, UVC-irradiated cells treated with endonuclease T4 V (total damage of DNA); Photo_1, Photo_0.1 and Photo_0.02 are cells treated with 50 μI of 1, 0.1 and 0.02 mg/ml recombinant CPD photolyase.
Example 3—CPD-Repair Activity by Immunoassay
[0076] Immunochemical assay experiments, using monoclonal commercial antibodies that recognize CPDs (Table 3), also confirmed that the recombinant CPD-photolyase from Hymenobacter sp. UV11 was purified as an active DNA repairing enzyme. In these experiments, calf-thymus DNA that contains single stranded DNA, was used as substrate and the recombinant CPD-photolyase reduced the presence CPDs completely. In this assay, UVC-irradiated and non-photolyase treated DNA were used as reference or control experiments. We detected repair activity even by using 1 μg ml of the recombinant photolyase, which means that the purified enzyme was diluted 5000 fold for this experiment. When experiments were carried out in darkness or when the recombinant enzyme was denatured by heating, we did not detect repair activity. These results also show that the recombinant CPD-photolyase from Hymenobacter sp. UV11 has been purified as an active enzyme which requires light for its repair activity.
[0077] Results were supported by the statistic test One-way ANOVA (p<0.05).
TABLE-US-00003 TABLE 3 Standard Mean error for the Test details Mean 1 Mean 2 Difference difference Significance Summary C+ vs. C− 2.348 0.1655 2.182 0.03399 Yes **** C+ vs. 2.348 0.26 2.088 0.03399 Yes **** Photo_2 + BL C+ vs. 2.348 0.2475 2.1 0.03399 Yes **** Photo_1.5 + BL C+ vs. 2.348 0.266 2.082 0.03399 Yes **** Photo_1 + BL C+ vs. 2.348 2.351 −0.003 0.03399 No ns Photo_1.5 Heat C+ vs. 2.348 2.197 0.151 0.03399 Yes * Photo_1.5 C+ vs. 2.348 2.251 0.097 0.03399 No ns Photo_1 C− vs. 0.1655 0.26 −0.0945 0.03399 No ns Photo_2 + BL C− vs. 0.1655 0.2475 −0.082 0.03399 No ns Photo_1.5 + BL C− vs. 0.1655 0.266 −0.1005 0.03399 No ns Photo_1 + BL C− vs. 0.1655 2.351 −2.185 0.03399 Yes **** Photo_1.5 Heat C− vs. 0.1655 2.197 −2.031 0.03399 Yes **** Photo_1.5 C− vs. 0.1655 2.251 −2.085 0.03399 Yes **** Photo_1 Photo_2 + BL vs. 0.26 0.2475 0.0125 0.03399 No ns Photo_1.5 + BL Photo_2 + BL vs. 0.26 0.266 −0.006 0.03399 No ns Photo_1.5 + BL Photo_2 + BL 0.26 2.351 −2.091 0.03399 Yes **** vs. Photo_1.5 Heat Photo_2 + BL 0.26 2.197 −1.937 0.03399 Yes **** vs. Photo_1.5 Photo_2 + BL 0.26 2.251 −1.991 0.03399 Yes **** vs. Photo_1 Photo_1.5 + BL 0.2475 0.266 −0.0185 0.03399 No ns vs. Photo_1 + BL Photo_1.5 + BL 0.2475 2.351 −2.103 0.03399 Yes **** vs. Photo 1.5 Heat Photo_1.5 + BL 0.2475 2.197 −1.949 0.03399 Yes **** vs . Photo_1.5 Photo_1.5 + BL 0.2475 2.251 −2.003 0.03399 Yes **** vs. Photo_1 Photo_1 + BL 0.266 2.351 −2.085 0.03399 Yes **** vs. Photo_1.5 Heat Photo_1 + BL 0.266 2.197 −1.931 0.03399 Yes **** vs. Photo_1.5 Photo_1 + BL 0.266 2.251 −1.985 0.03399 Yes **** vs. Photo_1 Photo_1.5 2.351 2.197 0.154 0.03399 Yes * Heat vs. Photo_1.5 Photo_1.5 2.351 2.251 0.1 0.03399 No ns Heat vs. Photo_1 Photo_1.5 2.197 2.251 −0.054 0.03399 No ns vs. Photo_1 Abbreviations are as follows: C−, non-irradiated calf thymus DNA (non-damaged DNA); C+, 10 Joules UVC-irradiated DNA (damaged DNA); Photo_1 + BL, Photo_1.5 + BL and Photo_2 + BL are UVC-irradiated DNA treated with 1, 1.5 and 2 μg/ml recombinant CPD-photolyase and blue light (BL); Photo_1.5 and Photo_1 are UVC-irradiated DNA treated with 1.5 and 1 μg/ml recombinant CPD-photolyase in darkness conditions, respectively; Photo_1.5 Heat is UVC-irradiated DNA treated with heat-inactivated recombinant CPD-photolyase (boiled for 10 min).
Example 4—Experiments Using the Recombinant 6,4-Bifunctional Photolyase as Repairing Enzyme
[0078] Immunochemical assays, using monoclonal commercial antibodies that recognize 6,4-photoproducts (Table 4) and CPDs (Table 5), also confirmed that the recombinant 6,4-bifunctional photolyase from Sphingomonas sp. UV9 was purified as an active DNA repairing enzyme. In these experiments, we detected repair activity of both DNA lesions CPD and 6,4-photoproducts, even by using 1 μg ml.sup.−1 of the recombinant 6,4-bifunctional photolyase.
[0079] These results Show that the recombinant 6,4-bifunctional photolyase from Sphingomonas sp. UV9 has been purified as an active enzyme and that this enzyme might repair both DNA lesions (CPD and 6,4-photoproducts).
[0080] 6,4-Photoproduct-Repair Activity by Immunoassay
[0081] Experiments using the recombinant 6,4-bifunctional photolyase from Sphingomonas sp. UV9 as repairing enzyme. immunodetection of 6,4-photoproducts. Results were supported by the statistic test One-way ANOVA (p<0.05).
TABLE-US-00004 TABLE 4 Standard Mean error for the Test details Mean 1 Mean 2 Difference difference Significance Summary C+ vs. C− 2.348 0.1705 2.177 0.02328 Yes **** C+ vs. 6.4 2.348 0.1925 2.155 0.02328 Yes **** C− vs. 6.4 0.1705 0.1925 −0.022 0.02328 No ns Abbreviations are as follows: C−, non-irradiated calf thymus DNA (non-damaged DNA); C+, 10 Joules UVC-irradiated DNA (damaged DNA); 6.4, DNA samples treated with the recombinant 6,4-bifunctional photolyase at a concentration of 2 mg/ml, as described above.
[0082] The CPD-Repair Activity by Immunoassay
[0083] Experiments using the recombinant 6,4-bi functional photolyase from Sphingomonas sp. UV9 as repairing enzyme. Immunodetection of CPD-photoproducts. Results were supported by the statistic test One-way ANOVA (p<0.05).
TABLE-US-00005 TABLE 5 Standard Mean error for the Test details Mean 1 Mean 2 Difference difference Significance Summary C+ vs. C− 2.14 0.1655 1.974 0.1641 Yes *** C+ vs. 6.4 2.14 0.4565 1.683 0.1641 Yes *** C− vs. 6.4 0.1655 0.4565 −0.291 0.1894 No ns Abbreviations are as follows: C−, non-irradiated calf thymus DNA (non-damaged DNA); C+, 10 Joules UVC-irradiated DNA (damaged DNA); 6.4, DNA samples treated with the recombinant 6,4-bifunctional photolyase at a concentration of 2 mg/ml, as described above.
Example 5—Method for the Qualitative Determination of CPD-Photolyase Activity
[0084] With the aim of fully CPDs formation, the plasmid pUC13-2 was UVC-irradiated (1000 J m, 254 nm) using a Spectroline lamp (model ENF-260C/FE). The full radiation was evaluated by MseI digestion (1 μg of plasmid-DNA was digested with 1 Unit of the restriction enzyme for 1 h at 37° C., in a total reaction volume of 50 μl) and 1% agarose gel electrophoresis at room temperature, for 1 h at 60V, using TAE as running buffer (TAE, 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA). DNA-restriction fragments were visualized by staining with ethidium bromide as follows: the gel was submerged in 0.5 μg/ml ethidium bromide for 30 min, washed with distilled water and visualized under UV light. DNA-damage was evidenced by the absence of the 780 kbp DNA fragment corresponding to the miniTn5 cassette. CPD-activity was evidenced by MseI digestion of UVC-irradiated pUC18-2 previously incubated with the CPD-photolyase at 1 μg/ml final concentration, at room temperature and blue light radiation, for 5 min for photoreduction and photorepair enzyme reactivation. The change in the electrophoretic profile of the MseI digested UVC-irradiated pUC18-2 plasmid as shown in
Example 6—Experiments Using the Recombinant 6,4-Photolyase as Repairing Enzyme
[0085] Immunochemical assays, using monoclonal commercial antibodies that recognize 6,4-photoproducts (Table 6) confirmed that the recombinant 6,4-photolyase from Sphingomonas sp. UV9 was purified as an active DNA repairing enzyme. These results show that the recombinant 6,4-photolyase from Sphingomonas sp. UV9 has been purified as an active enzyme and that 6,4-photoproducts.
[0086] 6,4-Photoproduct-Repair Activity by Immunoassay
[0087] Experiments using the recombinant 6,4-photolyase from Sphingomonas sp. UV9 as repairing enzyme. Immunodetection of 6,4-photoproducts. Results were supported by the statistic test One-way ANOVA (p<0.05).
TABLE-US-00006 TABLE 6 Standard Mean error for the Test details Mean 1 Mean 2 Difference difference Significance Summary C+ vs. C− 0.4530 0.1434 0.3096 0.02192 Yes **** C+ vs. 6.4 0.4530 0.1871 0.2659 0.02192 Yes **** C− vs. 6.4 0.1871 0.1434 0.0437 0.02192 Mo ns Abbreviations are as follows: C−, non-irradiated calf thymus DNA (non-damaged DNA); C+, 10 Joules UVC-irradiated DNA (damaged DNA); 6.4, DNA samples treated with the recombinart 6,4-photolyase at a concentration of 2 mg/ml, as described above.