Catalysis of DNA cleavage and nucleoside triphosphate synthesis by NM23-H2/NDP kinase share an active site that implies a DNA repair function.
NM23/NDP kinases play an important role in development and cancer but their biological function is unknown, despite an intriguing collection of biochemical properties including nucleoside-diphosphate kinase (NDP kinase), DNA binding and transcription, a mutator function, and cleavage of unusually structured DNA by means of a covalent enzyme-DNA complex. To assess the role of the nuclease in human NM23-H2, we sought to identify the amino acid responsible for covalent catalysis. By sequencing a DNA-linked peptide and by site-directed mutagenesis, we identified lysine-12, a phylogenetically conserved residue, as the amino acid forming the covalent complex with DNA. In particular, the epsilon-amino group acts as the critical nucleophile, because substitution with glutamine but not arginine completely abrogated covalent adduct formation and DNA cleavage, whereas the DNA-binding properties remained intact. These findings and chemical modification data suggest that phosphodiester-bond cleavage occurs by a DNA glycosylase/lyase-like mechanism known as the signature of base excision DNA repair nucleases. Involvement of NM23/NDP kinase in a DNA repair pathway would be consistent with its role in normal and tumor cell development. Additionally, lysine-12, which is known in the x-ray crystallographic structure to lie in the catalytic pocket involved in the NDP kinase phosphorylation reaction, was found essential also for the NDP kinase activity of NM23-H2, suggesting that the two catalytic activities of NM23-H2 are fundamentally connected.
Postel
E H
EH
Department of Molecular Biology, Princeton University, Princeton, NJ 08546-5414, USA. epostel@molbio.princeton.edu
Abramczyk
B M
BM
Levit
M N
MN
Kyin
S
S
D001665:Binding Sites; D001894:Borohydrides; D002384:Catalysis; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D008239:Lysine; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase / Q000378:metabolism* / Q000502:physiology; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism* / Q000502:physiology
D001894:Borohydrides; D054778:NM23 Nucleoside Diphosphate Kinases; D014157:Transcription Factors; D004247:DNA; C009282:sodium cyanoborohydride; C517938:NME1 protein, human; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins; D008239:Lysine