Characterization of the 3' → 5' exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues

Nucleoside diphosphate kinases (NDKs) have been characterized as a large family of conserved proteins that synthesize nucleoside triphosphates from nucleoside diphosphates and ATP (1). One of the main functions of the NDKs is the maintenance of the intracellular nucleoside triphosphate pools. However, it has been shown that the disruption of the single NDK genes in E. coli, Saccharomyces cerevisiae, and Schizosaccharomyces pombe has no effect on cell growth or morphology (2-4). Some microorganisms such as mycoplasma species and Thermotoga maritima lack an NDK gene altogether (1). E. coli strains with a disrupted NDK gene show a mutator phenotype which is thought to be due to imbalances in nucleotide pools (4, 5). It has become recognized that some NDK proteins have additional or different roles, in particular in mammals. These genes and their corresponding proteins are referred to here as NDKs, but the mammalian homologues have also been named NM23-H or NME genes based on the first discovery of a mouse gene that showed strongly reduced expression in metastatic mouse melanoma cell lines (NM23 stands for “nonmetastatic clone 23”) (6). To date, eight human NDK homologues have been identified (1). NDK1 (NM23-H1) seems to be involved in suppression of tumor metastasis since low expression of NDK1 has been linked to increased metastatic potential in many cell lines and tumors (6-9). The NDK proteins are usually expressed ubiquitously but NDK5, NDK7, and NDK8 are mainly found in testis (1, 10, 11). NDK4 and NDK6 are localized predominantly in mitochondria (12, 13). There is increasing evidence that some NDK proteins have DNA processing functions. In particular, NDK2 has been shown to be involved in regulation of transcription, by binding to and activating a nuclease-hypersensitive element in the c-myc promoter (14). Certain NDK proteins, such as NDK1, NDK2, and E. coli NDK can cleave DNA sequences with unusual structures in vitro (15-17). A recent study has shown that NDK1 is involved as the DNA cleavage component in a complex that promotes cytotoxic T-lymhocyte-mediated apoptosis (18) and this protein has been characterized biochemically as a 3' to 5' exonuclease (19). Recently, it has been reported that E. coli NDK possesses multifunctional base excision repair activities, namely uracil-DNA glycosylase, AP-lyase and 3'-phosphodiesterase activity, in vitro (20). However, subsequently, two different groups have reported that E. coli NDK does not possess uracil-DNA glycosylase (21, 22) and AP-lyase activity (21). Here, we have characterized the NDP kinase activity and DNA processing functions of eight human proteins that contain at least one domain homologous to E. coli NDK. We report that only human NDK1, NDK2 and NDK4 contain kinase activity and that human NDK1, NDK5, NDK7 and NDK8 retain 3' to 5' exonuclease activity.