title: Mutations in the nucleotide binding and hydrolysis domains of Helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function.
abstract: BACKGROUND
  Helicobacter pylori MutS2 (HpMutS2), an inhibitor of recombination during transformation is a non-specific nuclease with two catalytic sites, both of which are essential for its anti-recombinase activity. Although HpMutS2 belongs to a highly conserved family of ABC transporter ATPases, the role of its ATP binding and hydrolysis activities remains elusive.
  
  
  RESULTS
  To explore the putative role of ATP binding and hydrolysis activities of HpMutS2 we specifically generated point mutations in the nucleotide-binding Walker-A (HpMutS2-G338R) and hydrolysis Walker-B (HpMutS2-E413A) domains of the protein. Compared to wild-type protein, HpMutS2-G338R exhibited ~2.5-fold lower affinity for both ATP and ADP while ATP hydrolysis was reduced by ~3-fold. Nucleotide binding efficiencies of HpMutS2-E413A were not significantly altered; however the ATP hydrolysis was reduced by ~10-fold. Although mutations in the Walker-A and Walker-B motifs of HpMutS2 only partially reduced its ability to bind and hydrolyze ATP, we demonstrate that these mutants not only exhibited alterations in the conformation, DNA binding and nuclease activities of the protein but failed to complement the hyper-recombinant phenotype displayed by mutS2-disrupted strain of H. pylori. In addition, we show that the nucleotide cofactor modulates the conformation, DNA binding and nuclease activities of HpMutS2.
  
  
  CONCLUSIONS
  These data describe a strong crosstalk between the ATPase, DNA binding, and nuclease activities of HpMutS2. Furthermore these data show that both, ATP binding and hydrolysis activities of HpMutS2 are essential for the in vivo anti-recombinase function of the protein.
authors: Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India.
authors: Prashant P
authors: PP
authors: Damke
authors: Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India.
authors: Rajkumar
authors: R
authors: Dhanaraju
authors: CEA, Institute of Cellular and Molecular Radiobiology, Fontenay aux Roses, France.|INSERM UMR967, Fontenay aux Roses, France.|Universités Paris Diderot et Paris Sud, Fontenay aux Roses, France.
authors: Stéphanie
authors: S
authors: Marsin
authors: CEA, Institute of Cellular and Molecular Radiobiology, Fontenay aux Roses, France. pablo.radicella@cea.fr.|INSERM UMR967, Fontenay aux Roses, France. pablo.radicella@cea.fr.|Universités Paris Diderot et Paris Sud, Fontenay aux Roses, France. pablo.radicella@cea.fr.
authors: J Pablo
authors: JP
authors: Radicella
authors: Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India. dnrao@biochem.iisc.ernet.in.
authors: Desirazu N
authors: DN
authors: Rao
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016480:Helicobacter pylori / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic
chemical_list: D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D051719:MutS DNA Mismatch-Binding Protein