Abstract
Photolyases are flavoproteins that repair ultraviolet-induced DNA lesions (cyclobutane pyrimidine dimer or CPD, and pyrimidine (6-4) pyrimidone photoproducts or (6-4)-PPs), using blue light as an energy source. These enzymes are substrate specific, meaning that a specific photolyase repairs either a CPD or a (6-4)-PP. In this work, we produced a class II CPD-photolyase (called as PhrSph98) from the Antarctic bacterium Sphingomonas sp. UV9 by recombinant DNA technology and we purified the enzyme using immobilized metal affinity chromatography. By using an immunochemistry assay, with monoclonal antibodies against CPD and (6-4)-PP, we found that PhrSph98 repairs both DNA lesions. The result was confirmed by immunocytochemistry using immortalized non-tumorigenic human keratinocytes. Results from structure prediction, pocket computation, and molecular docking analyses showed that PhrSph98 has the two expected protein domains (light-harvesting antenna and a catalytic domain), a larger catalytic site as compared with photolyases produced by mesophilic organisms, and that both substrates fit the catalytic domain. The results obtained from predicted homology modeling suggest that the electron transfer pathway may occur following this pathway: Y389-W369-W390-F376-W381/FAD. The evolutionary reconstruction of PhrSph98 suggests that this is a missing link that reflects the transition of (6-4)-PP repair into the CPD repair ability for the class II CPD-photolyases. To the best of our knowledge, this is the first report of a naturally occurring bifunctional, CPD and (6-4)-PP, repairing enzyme.
Key points
• We report the first described bifunctional CPD/(6-4)-photoproducts repairing enzyme. The bifunctional enzyme reaches the nuclei of keratinocyte and repairs the UV-induced DNA damage. The enzyme should be a missing link from an evolutionary point of view. The enzyme may have potential uses in the pharmaceutical and cosmetic industries.
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Acknowledgments
The authors thank the Uruguayan Antarctic Institute for the logistic support during the stay in the Antarctic Base Artigas. S. Castro-Sowinski, L. Canclini, and J. J. Marizcurrena are members of the National Research System (SNI, Sistema Nacional de Investigadores).
Funding
This work was partially supported by PEDECIBA (Programa de Desarrollo de las Ciencias Básicas), CSIC (Project C667), ANII (Project FMV_3_2016_1_1226654), Comisón Honoraria de Lucha Contra el Cáncer, and donations by Celsius Laboratory (http://www.celsius.uy/). The work of JJM was supported by ANII and CAP (Comisión Académica de Posgrado, UdelaR). The provisional patent was funded by ANII (Agencia Nacional de Investigación e Innovación, PAT_X_2017_1_140739) & CSIC (Comisión Sectorial de Investigación Científica).
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JJM produced and purified the recombinant photolyase and performed the in vitro DNA photorepair, the structure prediction, pocket computation, and molecular docking experiments. AC, LC, and PH conducted the immunocytochemistry assays. DV conducted the HPLC experiments. TL conducted the evolutionary reconstruction. SCS guided most experiments and wrote the manuscript.
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Marizcurrena, J.J., Acosta, S., Canclini, L. et al. A natural occurring bifunctional CPD/(6-4)-photolyase from the Antarctic bacterium Sphingomonas sp. UV9. Appl Microbiol Biotechnol 104, 7037–7050 (2020). https://doi.org/10.1007/s00253-020-10734-5
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DOI: https://doi.org/10.1007/s00253-020-10734-5