title: Characterization of an ATP-regulated DNA-processing enzyme and thermotolerant phosphoesterase in the radioresistant bacterium Deinococcus radiodurans.
abstract: 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.
authors.affiliation: Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India.
authors.forename: Swathi
authors.initials: S
authors.lastname: Kota
authors.forename: C Vijaya
authors.initials: CV
authors.lastname: Kumar
authors.forename: Hari S
authors.initials: HS
authors.lastname: Misra
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D034301:Deinococcus / Q000201:enzymology* / Q000378:metabolism; D005720:Gamma Rays; D009708:Nucleotidases / Q000737:chemistry / Q000378:metabolism*; D011836:Radiation Tolerance
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D009708:Nucleotidases; D045643:DNA Repair Enzymes