A multiprotein DNA-processing complex identified from Deinococcus radiodurans exhibits uncharacterized ATP-sensitive nuclease functions. DR0505 was one of the 24 polypeptides identified from the complex. It contains two 5' nucleotidase motifs, one is at the C-terminal end of the N-terminal CPDD (calcineurin phosphodiesterase domain), with the second at the C-terminal end of the protein. Recombinant DR0505 showed both phosphomonoesterase and phosphodiesterase activities with chromogenic substrates, showing higher affinity for bis-(p-nitrophenyl) phosphate than for p-nitrophenyl phosphate. The enzyme exhibited pH optima ranging from 8.0 to 9.0 and metal-ion-dependent thermotolerance of esterase functions. Both mono- and di-esterase activities were stable at temperatures up to 50°C in the presence of Mg²⁺, whereas monoesterase activity was observed at temperatures up to 80°C in the presence of Mn²⁺ and up to 50°C with Ca²⁺. The purified enzyme showed 5' nucleotidase activity on a wide range of natural mononucleotides including cyclic mononucleotides and 8-oxo-GMP. DR0505 showed a nearly 7-fold higher activity on ADP than AMP, but this activity was inhibited with ATP. Interestingly, DR0505 also showed single-stranded endonuclease and 3'®5' exonuclease activities on both single-stranded and double-stranded DNA-substrates. Unlike for the exonuclease activity, the single-stranded endonuclease activities observed on stem-loop substrates and at the single strand-double-strand junction in forked-hairpin substrates were not inhibited with ATP. These results suggested that DR0505 is an ATP-regulated DNA-processing enzyme and a thermotolerant esterase in vitro. We therefore suggest possible roles of this enzyme in nucleotide recycling and DNA processing, which is required for efficient double-strand break repair and the high radiation tolerance observed in D. radiodurans.