THE SUN EMITS NEAR-UV AND UV LIGHT ENGENDERING DELETERIOUS PHOTOCHEMICAL REACTIONS. HOWEVER HEREDITY IS IMPOSSIBLE WITHOUT GENETIC CONTINUITY LEADING TO THE EVOLUTION OF BACTERIAL DNA REPAIR SYSTEMS. WE PROPOSE TO EXTEND OUR COMPARATIVE ANALYSIS OF EXTREMOPHILE DNA PHOTOLYASES FROM THE ARCHAEON HYPERTHERMOPHILE S. SOLFATARICUS (SSPL) AND THE BACTERIAL PSYCHROPHILE C. PSYCHRERYTHRAEA (CPPL). THIS IS A SUCCESSOR REQUEST FOR NNX13AH33G. THE RESEARCH TEAM OF ROBERT STANLEY (TEMPLE) YVONNE GINDT (MONTCLAIR STATE) AND DAVID BERATAN (DUKE) WILL REMAIN THE SAME. KEY OBJECTIVES: I. COMPARE AND CONTRAST MESOPHILIC LIGHT-DRIVEN ENZYMATIC DNA REPAIR WITH EXTREMOPHILIC ENZYMES EXTENDING OUR EARLY WORK TO UNDERSTAND HOW BIOPHYSICAL PROCESSES ARE ADAPTED TO EXTREME ENVIRONMENTAL CONDITIONS. II. EXPLORE PSYCHROPHILE SUBSTRATE PROMISCUITY AS A SOURCE OF GREATER BIOSYNTHETIC COMPLEXITY WITH A FOCUS ON FLAVIN ADENINE DINUCLEOTIDE AND ITS ANALOGS. III. TEST THE HYPOTHESIS THAT MODERN PLS EVOLVED FROM AN UR-PL WHOSE KERNEL CONTAINS ONLY THE FAD COFACTOR IN A SINGLE DOMAIN PROTEIN. A FEW HIGHLIGHTS FROM THE PREVIOUS PERIOD INCLUDE: THE DISCOVERY THAT ARCHAEAL HYPERTHERMOPHILIC SSPL IS A MISSING LINK IN THE EVOLUTION OF PHOTOLYASES INTO THE BLUE-LIGHT PHOTOSENSORY CRYPTOCHROME (CRY) PROTEIN. EUKARYOTIC CRY PERFORMS SEVERAL LIGHT-DRIVEN FUNCTIONS DEPENDING ON THE ORGANISM (PHOTOMOTILITY IN PLANTS ETC.). THE DISCOVERY OF COFACTOR PROMISCUITY IN PSYCHROPHILIC CPPL. PLS CONTAIN A SECOND COFACTOR THAT ACTS AS A LIGHT ANTENNA TO REDUCE THE CONSERVED CATALYTIC FLAVIN ADENINE DINUCLEOTIDE (FAD) MOLECULE. CPPL CAN TAKE UP EITHER A FOLATE OR A DIFFERENT FLAVIN (FMN OR FLAVIN MONONUCLEOTIDE). THE TEMPERATURE DEPENDENCE OF SUBSTRATE BINDING AND OF DNA REPAIR HAVE BEEN MEASURED IN MESOPHILIC AND HYPERTHERMOPHILIC PLS FROM NEAR FREEZING UP TO THE DENATURATION POINT OF THE PROTEIN. WHILE THE FREE ENERGY OF SUBSTRATE BINDING APPEARS INDEPENDENT OF TEMPERATURE SSPL SHOWS SIGNIFICANTLY FASTER REPAIR AT ALL TEMPERATURES. RELEVANCE TO NASA EXOBIOLOGY MISSION GOALS: THIS PROPOSAL RESPONDS TO SPECIFIC MISSION GOALS OF NASA EXOBIOLOGY IN APPENDIX C.5 OF THE 2016 ROSES SOLICITATION. OUR RESEARCH GOALS ADDRESS AIMS (III) AND (V) FROM EARLY EVOLUTION OF LIFE AND THE BIOSPHERE (EELB). THESE INCLUDE EELB(III) - DETERMINE THE ORIGINAL NATURE OF BIOLOGICAL ENERGY TRANSDUCTION TO TEST HYPOTHESES REGARDING THE ORIGINAL NATURE OF KEY BIOLOGICAL PROCESSES AND EELB(V) - INVESTIGATE THE EVOLUTION OF GENES PATHWAYS AND MICROBIAL SPECIES SUBJECT TO LONGTERM ENVIRONMENTAL CHANGE RELEVANT TO THE ORIGIN OF LIFE ON EARTH AND THE SEARCH FOR LIFE ELSEWHERE . APPROACH AND METHODOLOGY: TO EXTEND THESE STUDIES WE WILL PERFORM COMPARATIVE ANALYSES OF PHOTOBIOLOGICAL ENERGY TRANSDUCTION PROTEIN:SUBSTRATE BINDING RELEASE AND BIOLOGICAL ELECTRON TRANSFER IN EXTREMOPHILE DNA PHOTOLYASES. PHOTOLYASE ENCOMPASSES ALL BASIC AND CRITICAL BIOCHEMICAL PROCESSES BUT HAS THE ADVANTAGE OF BEING LIGHT-DRIVEN. THIS AFFORDS A SEPARATION OF ENZYMATIC EVENTS SO THAT THE EFFECTS OF EXTREME ENVIRONMENTAL CHANGES CAN BE ACCURATELY MONITORED. EXPERIMENTS WILL BE PERFORMED BY THE STANLEY GROUP USING ULTRAFAST LASER SPECTROSCOPY AND COMPLEMENTED BY QM/MM SIMULATIONS BY THE BERATAN GROUP. STANLEY AND GINDT WILL EXECUTE SUBSTRATE BINDING/REPAIR EXPERIMENTS OVER A WIDER RANGE OF ENVIRONMENTAL CONDITIONS INCLUDING TEMPERATURE VISCOSITY AND IONIC STRENGTH TO TEST THE FUNCTIONAL LIMITS TO ENVIRONMENTAL EXTREMES. GINDT WILL PROVIDE THERMODYNAMIC AND ELECTROCHEMICAL MEASUREMENTS TO COMPLEMENT KINETICS DATA TAKEN BY THE STANLEY GROUP. AN IMPORTANT COMPLEMENTARY AIM OF THIS PROPOSAL IS THE STUDY OF UV-DNA DAMAGE UNDER EXTREME ENVIRONMENTAL CONDITIONS WHICH WILL INVOLVE EFFORT FROM THE RESEARCH TEAM AS A WHOLE.