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Characterization of a cold-adapted DNA photolyase from C. psychrerythraea 34H

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Abstract

The phrB gene encoding a putative cold-adapted DNA photolyase was cloned from the bacterial genomic DNA of Colwellia psychrerythraea 34H, a psychrophilic bacterium. Recombinant DNA photolyase, rCpPL, was overexpressed and purified from three different vectors. rCpPL binds its DNA substrate by flipping a cyclobutane pyrimidine dimer (CPD) into its active site and repairs CPD-containing DNA in vitro. rCpPL contains one catalytic flavin adenine dinucleotide (FAD) cofactor, but displays promiscuity in cofactor binding, in which either a flavin mononucleotide (FMN) or a methenyltetrahydrofolate (MTHF) molecule is bound as an antenna molecule and found in sub-stoichiometric amounts. The UV/Vis spectrum of oxidized rCpPL shows that the FADOX absorption maximum is the most red-shifted reported for a PL, suggesting a unique cavity electrostatic environment. Modest FAD vibronic structure suggests that the binding pocket is more flexible than warmer PLs, corroborating the hypothesis that psychrophilic proteins must be highly flexible to function at low temperatures. Fluorescence excitation data show that the freshly purified flavin cofactor is in its fully reduced state (FADH¯). A homology analysis of PL protein structures spanning 70 °C in growth temperature supports the data that the structure of CpPL is quite different from its warmer cousins.

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Acknowledgements

We wish to thank Mark Olsen and Cephalon for the donation of the Agilent HPLC. We also want to acknowledge Dr. Georges Feller, Dr. Aurora Martinez, Dr. Yvonne Gindt, and Dr. Peter S. Kessler for helpful discussions. The AnPL expression plasmid was a generous gift of Prof. A. Sancar, UNC Chapel Hill. S.M. and R.J.S. acknowledge support from NASA Exobiology Grant NNX13AH33G. This research was supported in part by the NSF Grant CHE-0847855. A.R. received support from NSF-REU supplement to CHE-0847855.

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  1. Ananthi Rajamoorthi

    Present address: Department of Biochemistry, Saint Louis University School of Medicine, 3854 McDonald Ave, St. Louis, MO, 63116, USA

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  1. Department of Chemistry, Temple University, Philadelphia, PA, 19122, USA

    Sudipto Munshi, Ananthi Rajamoorthi & Robert J. Stanley

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Communicated by L. Huang.

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792_2017_953_MOESM1_ESM.docx

Supplementary material 1 The SI includes buffer recipes, cloning, purification and harvesting protocols, photoreduction, and repair assay information. SI Figs. 1–11 include relative disorder probabilities and 3D homology structures for other PLs not discussed in the main text, HPLC analyses of cofactors and repair, absorption spectra, and SDS-PAGE analysis of the rCpPL-MBP fusion and rCpPL(S) system. Table SI-1 includes a calculation of FMN cofactor content (DOCX 11008 kb)

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Munshi, S., Rajamoorthi, A. & Stanley, R.J. Characterization of a cold-adapted DNA photolyase from C. psychrerythraea 34H. Extremophiles 21, 919–932 (2017). https://doi.org/10.1007/s00792-017-0953-z

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