Extremophile DNA Photolyases: DNA Repair under Extreme Conditions

Abstract

Adaptation to extreme environments is a seminal characteristic of life on Earth. Nowhere is this property more strongly evident than in single-celled organisms. Bacteria, archaea, and eukaryotes all have representatives that thrive in cold or hot environments. Many of these extremophile enzymes share significant amino acid sequence homology in spite of very different optimal growth temperatures (TG). Based on a simple amino acid sequence analysis it is unclear what adaptive changes are required to tailor homologous enzymes to function in extreme environments.A case in point is DNA photolyase (PL), a monomeric flavoprotein that binds UV-damaged DNA and repairs it by blue light activated picosecond electron transfer from a conserved flavin adenine dinucleotide cofactor (FADH¯) to the tightly bound cyclobutylpyrimidine dimer DNA lesion. These enzymes also include a second light-harvesting cofactor. Here we present a comparative analysis of three recombinant CPD photolyases, hyperthermophilic Sulfolobus solfataricus (TG=353K, rSsPL), mesophilic E. coli (TG=310K, rEcPL), and psychrophilic Colwellia psychrerythraea (TG=281K, rCpPL).All PLs utilize FADH¯ bound in a highly conserved site for repair. In addition, each PL demonstrates different properties for its 2nd cofactor. We used a variety of biochemical, physical, and molecular biological tools to compare these extremophile proteins with regard to repair yield, cofactor reduction potential and excited state properties. The difference in the stability of the redox state of the purified protein suggests different structural adaptations of each PL to their respective thermal environments. Denaturation studies reveal that rSsPL has a very stable structure, whereas rCpPL is extremely sensitive to its thermal and aqueous environments. A comparison of cofactor absorption and emission spectra reveal significant differences in how the flavin cofactor is bound in the binding pocket, in spite of significant amino acid sequence homology for this cavity across all proteins.