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First characterisation of a CPD-class I photolyase from a UV-resistant extremophile isolated from High-Altitude Andean Lakes

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Abstract

UV-resistant Acinetobacter sp. Ver3 isolated from High-Altitude Andean Lakes (HAAL) in Argentinean Puna, one of the highest UV exposed ecosystems on Earth, showed efficient DNA photorepairing ability, coupled to highly efficient antioxidant enzyme activities in response to UV-B stress. We herein present the cloning, expression, and functional characterization of a cyclobutane pyrimidine dimer (CPD)-class I photolyase (Ver3Phr) from this extremophile to prove its involvement in the previously noted survival capability. Spectroscopy of the overexpressed and purified protein identified flavin adenine dinucleotide (FAD) and 5,10-methenyltetrahydrofolate (MTHF) as chromophore and antenna molecules, respectively. All functional analyses were performed in parallel with the ortholog E. coli photolyase. Whereas the E. coli enzyme showed the FAD chromophore as a mixture of oxidised and reduced states, the Ver3 chromophore always remained partly (including the semiquinone state) or fully reduced under all experimental conditions tested. Functional complementation of Ver3Phr in Phr−-RecA E. coli strains was assessed by traditional UFC counting and measurement of DNA bipyrimidine photoproducts by HPLC coupled with electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) detection. The results identified strong photoreactivation ability in vivo of Ver3Phr while its nonphotoreactivation function, probably related with the stimulation of nucleotide excision repair (NER), was not as manifest as for EcPhr. Whether this is a question of the approach using an exogenous photolyase incorporated in a non-genuine host or a fundamental different behaviour of a novel enzyme from an exotic environment will need further studies.

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Authors and Affiliations

  1. Planta Piloto de Procesos Industriales y Microbiológicos (PROIMI), CCT, CONICET, Av. Belgrano y Pasaje Caseros, 4000, S. M. de Tucumán, Argentina

    Virginia Helena Albarracín & María Eugenia Farias

  2. Facultad de Ciencias Naturales e Instituto Miguel Lillo, Universidad Nacional de Tucumán, 4000, Tucumán, Argentina

    Virginia Helena Albarracín

  3. Max-Planck-Institute for Chemical Energy Conversion, Stiftstrasse 34-36, D-45470, Mülheim, Germany

    Virginia Helena Albarracín, Julian Simon, Gopal P. Pathak & Wolfgang Gärtner

  4. Laboratorio de Cinética y Fotoquímica. CITSE-CONICET, Facultad de Agronomía y Agroindustrias, Universidad de Santiago del Estero (UNSE), RN9 Km 1125, 4206, Santiago del Estero, Argentina

    Lorena Valle & Claudio Darío Borsarelli

  5. Laboratoire “Lésions des Acides Nucléiques” INaC/SCIB UMR-E3 CEA-UJF/CEA-Grenoble, 17 avenue des Martyrs, 38054, Grenoble Cedex 9, France

    Thierry Douki & Jean Cadet

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  1. Virginia Helena Albarracín
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  2. Julian Simon
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  3. Gopal P. Pathak
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  7. Claudio Darío Borsarelli
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Correspondence to Virginia Helena Albarracín or Wolfgang Gärtner.

Additional information

Electronic supplementary information (ESI) available: Table S2 and Fig. S1, S3 and S4. See DOI: 10.1039/c3pp50399b

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Albarracín, V.H., Simon, J., Pathak, G.P. et al. First characterisation of a CPD-class I photolyase from a UV-resistant extremophile isolated from High-Altitude Andean Lakes. Photochem Photobiol Sci 13, 739–751 (2014). https://doi.org/10.1039/c3pp50399b

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