Abstract
The NM23 gene family in humans is implicated in differentiation and cancer, but the biochemical mechanisms are unknown. Most NM23 proteins have phosphotransferase (nucleoside diphosphate kinase) activity, and the second human isoform, NM23-H2, also binds to a nuclease-hypersensitive c-MYC promoter element through which it activates c-MYC transcription. It is shown here that this DNA binding can result in double-stranded breaks. The DNA breaks occur within repeated sequence elements in the linear nuclease-hypersensitive duplex and leave staggered ends with 5-nucleotide-long 3'-extensions. The enzyme also cleaves supercoiled plasmid DNA to yield nicked circular and unit length linear products. The cleavage reaction requires only NM23-H2, DNA, Mg(2+), and buffer, occurs in the absence of denaturing conditions, and can be reversed by EDTA. The cleaved DNA strands have free 3'-OH groups, and protein is attached to the 5'-phosphoryl ends. Transfer of (32)P radioactivity from DNA to NM23-H2 has been observed, and a covalent polypeptide-DNA complex has been isolated and identified by Western blotting as NM23-H2. Since covalent protein-DNA complexes are known to serve the role of breaking and rejoining DNA strands, the present findings suggest that NM23-H2 is involved in DNA structural transactions necessary for the activity of the c-MYC promoter.
Highlights
The NM23 gene was originally identified as a potential metastasis suppressor gene by virtue of its reduced expression in highly metastatic melanoma and breast carcinoma cells [1,2,3]
When EDTA was added at the end of the cleavage reaction followed by proteinase K treatment, the cleavages were restored almost completely in both the uniquely end-labeled 3Ј- and 5Ј-end DNA fragments (Fig. 3C, lanes 4 versus 2 and 9 versus 7). These results demonstrated that 1) DNA breaks occur in both strands and 2) that the breaks can be restored upon removal of divalent cations by EDTA treatment; the latter suggests that the cleavage reaction is mediated by a covalent protein-DNA complex
Numerous observations in the past have caused speculation that the NM23/NDP kinase family of proteins may perform a more sophisticated role in cell physiology than merely catalysis of nonspecific phosphoryl group transfer [3, 19, 38]. Buttressing these ideas was our discovery that NM23-H2/NDP kinase-B is a DNA-binding and transcriptionally active protein [23, 39]
Summary
NM23-H2 was purified from overexpressing bacteria by ammonium sulfate fractionation and hydroxyapatite chromatography as described [23], with the addition of an ion exchange chromatography step prior to hydroxyapatite fractionation. The DEAE column retains the DNA and. Most bacterial proteins, including bacterial NDP kinase, whereas NM23-H2 is eluted with 50 mM Tris buffer, pH 8 [19]. Protein preparations were apparently homogeneous as assessed by SDS-PAGE and hexameric as determined by size exclusion chromatography [26]. Affinity purified NM23-H2 was prepared by applying the 60 –90% ammonium sulfate fraction to a Blue Sepharose [19] or Reactive Yellow agarose, column [27] and subsequent elution with a NaCl gradient. NM23-H1 was purified by ammonium sulfate fractionation (60 –90% saturation), elution from DEAE-cellulose with a NaCl gradient, followed by hydroxyapatite and size exclusion chromatography [23, 26]
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