Cloning, purification and biophysical characterization of extremophile DNA photolyases

Abstract

DNA photolyase (PL) is a flavoprotein that repairs UV light induced cyclobutylpyrimidine dimers (CPDs) in DNA by a photo-induced electron transfer (PET) mechanism. All our understanding about the structure and function of PL is derived from studying the mesophilic forms of the protein that have evolved to function at ambient temperature. However, the discovery of extremophiles that inhabit and thrive at thermally extreme environments on Earth has raised interesting questions about how PL functions in these organisms. Since CPD repair by PL occurs by a PET mechanism, according to Marcus theory for electron transfer, the rate constants for the ET process are expected to be exponentially sensitive to temperature, rendering the CPD repair process, as understood from studying mesophilic PLs, highly improbable at extreme temperatures. However, we have found that two extremophile PLs: a thermo-stable PL from the hyperthermophilic archaeon Sulfolobus solfataricus (SsPL) and a cold-adapted PL from the psychrophilic bacterium Colwellia psychrerythraea 34H (CpPL), are both fully competent to repair CPD-containing DNA. Thus, we hypothesize that these extremophile PLs have evolved adaptation features that are uniquely suited for function in their respective thermal environments. In order to understand how DNA repair by PL occurs at these thermal extremes, the genes for both SsPL and CpPL were cloned, the recombinant proteins were overexpressed, purified and their biophysical and DNA repair properties were characterized. Both PLs were found to contain a noncovalently bound catalytic FAD molecule, as in other PLs, but showed differences in the identity of their antenna chromophores. Unique spectral features of the cofactors, indicative of structural adaptations to their respective thermal environments were observed. CpPL was found to be extremely sensitive to its thermal and aqueous environments, whereas SsPL displayed extreme thermal stability. The formation of a unique anionic semiquinone radical state (FAD•−) was observed for the antenna cofactor-lacking mutant of SsPL, something that has never been observed in any true CPD PL characterized so far. A comparative analysis of the kinetics of temperature-dependent CPD repair by mesophilic and thermophilic PLs was performed and the results suggest the SsPL is indeed suited for CPD repair at high temperatures. Analysis of thermal denaturation and the temperature-dependent CPD repair studies also indicated the importance of the antenna cofactor in the thermophilic PLs. Finally, computational analysis of the structure of the extremophile PLs revealed unique features related to temperature adaptation. Thus, based on various experimental and computational approaches, an initial picture of how these extremophile PLs have adapted to their respective extreme thermal environments, was obtained.