Archaeal　NurA　protein　plays　a　key　role　in　producing　3′-single　stranded　DNA　used　for　homologous　recombination　repair,　together　with　HerA,　Mre11,　and　Rad50.　Herein,　we　describe　biochemical　characteristics　and　roles　of　key　amino　acid　residues　of　the　NurA　protein　from　the　hyperthermophilic　euryarchaeon　Thermococcus　barophilus　Ch5　(Tba-NurA).　Tba-NurA　possesses　5′–3′　exonuclease　activity　for　degrading　DNA,　displaying　maximum　efficiency　at　45　°C–65　°C　and　at　pH　8.0　in　the　presence　of　Mn2+.　The　thermostable　Tba-NurA　also　possesses　endonuclease　activity　capable　of　nicking　plasmid　DNA　and　circular　ssDNA.　Mutational　data　demonstrate　that　residue　D49　of　Tba-NurA　is　essential　for　exonuclease　activity　and　is　involved　in　binding　ssDNA　since　the　D49A　mutant　lacked　exonuclease　activity　and　reduced　ssDNA　binding.　The　R96A　and　R129A　mutants　had　no　detectable　dsDNA　binding,　suggesting　that　residues　R96　and　R129　are　important　for　binding　dsDNA.　The　abolished　degradation　activity　and　reduced　dsDNA　binding　of　the　D120A　mutant　suggest　that　residue　D120　is　essential　for　degradation　activity　and　dsDNA　binding.　Additionally,　residues　Y392　and　H400　are　important　for　exonuclease　activity　since　these　mutations　resulted　in　exonuclease　activity　loss.　To　our　knowledge,　it　is　the　first　report　on　biochemical　characterization　and　mutational　analysis　of　the　NurA　protein　from　Thermococcus.　©　2024　Institut　Pasteur