title: Characterization of the 3' --> 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues.
abstract: Nucleoside diphosphate kinases (NDKs), an evolutionarily conserved family of proteins, synthesize nucleoside triphosphates from nucleoside diphosphates and ATP. Here, we have characterized the kinase activity and DNA processing functions of eight human proteins that contain at least one domain homologous to Escherichia coli NDK. Not all human proteins with NDK-like domains exhibited NDK activity when expressed as recombinant proteins in E. coli. Human NDK1 (NM23-H1) has been reported to have 3' --> 5' exonuclease activity. In addition to human NDK1, we also find that human NDK5, NDK7, and NDK8 contain 3' --> 5' exonuclease activity. Site-directed mutagenesis, competition assays between wild-type and mutant NDK proteins, and NMR studies confirmed that the DNA-binding and 3' --> 5' exonuclease activity of human NDK1 is an intrinsic activity of the protein. Using double-stranded DNA substrates containing modified bases, human NDK1 efficiently excised nucleotides from the single-strand break produced by APE1 or Nth1. When human cells were treated with various DNA-damaging agents, human NDK1 translocated from the cytoplasm to the nucleus. These results suggest that, in addition to maintenance of nucleotide pool balance, the human NDK-like proteins may have previously unrecognized roles in DNA nucleolytic processing.
authors.affiliation: Division of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
authors.forename: Jung-Hoon
authors.initials: JH
authors.lastname: Yoon
authors.forename: Purnima
authors.initials: P
authors.lastname: Singh
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Lee
authors.forename: Junzhuan
authors.initials: J
authors.lastname: Qiu
authors.forename: Sheng
authors.initials: S
authors.lastname: Cai
authors.forename: Timothy R
authors.initials: TR
authors.lastname: O'Connor
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chen
authors.forename: Binghui
authors.initials: B
authors.lastname: Shen
authors.forename: Gerd P
authors.initials: GP
authors.lastname: Pfeifer
mesh_terms: D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D005092:Exonucleases / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification; D016415:Sequence Alignment
chemical_list: D054778:NM23 Nucleoside Diphosphate Kinases; D011994:Recombinant Proteins; C517938:NME1 protein, human; D009701:Nucleoside-Diphosphate Kinase; D005092:Exonucleases