Edith H. Postel

Human NM23/nucleoside diphosphate kinase regulates gene expression through DNA binding to nuclease-hypersensitive transcriptional elements.

NM23-H2/NDP kinase B has been identified as a sequence-specific DNA-binding protein withaffinity for a nuclease-hypersensitive element of the c-MYC gene promoter (Postel et al.,1993). The ability of Nm23-H2 to activate c-MYC transcription in vitro and in vivo via thesame element demonstrates the biological significance of this interaction. Mutational analyseshave identified Arg34, Asn69 and Lys135 as critical for DNA binding, but not required forthe NDP kinase reaction. However, the catalytically important His118 residue is dispensiblefor sequence-specific DNA binding, suggesting that sequence-specific DNA recognition andphosphoryl transfer are independent properties. Nm23-H2 also has an activity that cleavesDNA site-specifically, involving a covalent protein-DNA complex. In a DNAsequence-dependent manner, Nm23-H2 recognizes additional target genes for activation, includingmyeloperoxidase, CD11b, and CCR5, all involved in myeloid-specific differentiation. Moreover,both NM23-H1 and Nm23-H2 bind to nuclease hypersensitive elements in the platelet-derivedgrowth factor PDGF-A gene promoter sequence-specifically, correlating with either positiveor negative transcriptional regulation. These data support a model in which NM23/NDP kinasemodulates gene expression through DNA binding and subsequent structural transactions.

Multiple biochemical activities of NM23/NDP kinase in gene regulation.

NM23/NDPk proteins play critical roles in cancer and development; however, our understanding of the underlying biochemical mechanisms is still limited. This large family of highly conserved proteins are known to participate in many events related to DNA metabolism, including nucleotide binding and nucleoside triphosphate synthesis, DNA binding and transcription, and cleavage of DNA strands via covalent protein–DNA complexes. The chemistry of the DNA-cleavage reaction of NM23-H2/NDPk is characteristic of DNA repair enzymes. Both the DNA cleavage and the NDPk reactions are conserved between E. coli and the human enzymes, and several conserved amino acid side chains involved in catalysis are shared by these reactions. It is proposed here that NM23/NDP kinases are important regulators of gene expression during development and cancer via previously unrecognized roles in DNA repair and recombination, and via previously unrecognized pathways and mechanisms of genetic control.

NM23-NDP kinase

NM23 belongs to a large family of structurally and functionally conserved proteins consisting of 4-6 identically folded subunits of approximately 16-20 kDa. These oligomeric proteins exhibit nucleoside diphosphate kinase (NDPK) activity that catalyzes nonsubstrate specific conversions of nucleoside diphosphates to nucleoside triphosphates. Many NM23 proteins bind DNA. In vivo, NM23-NDPKs regulate a diverse array of cellular events including growth and development. They are also implicated in the pathogenesis and metastasis of tumors. The mechanism whereby NM23 regulates gene expression is proposed to entail DNA-binding and subsequent alterations in promoter DNA structure. Accordingly, NM23 has the potential to become a useful reagent for gene manipulations.

Cleavage of DNA by Human NM23-H2/Nucleoside Diphosphate Kinase Involves Formation of a Covalent Protein-DNA Complex

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, Mg2+, 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 32P 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.

The interaction of nucleoside diphosphate kinase B with Gβγ dimers controls heterotrimeric G protein function

Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein βγ dimer (Gβγ) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in Gβ1γ2 dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating Gαs and Gαi subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish Gβ1 orthologs, Gβ1 and Gβ1like, causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of Gβ1/Gβ1like is associated with a down-regulation in caveolins, AC-regulating Gα-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with Gβγ dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.

Interactions between Escherichia coli Nucleoside-diphosphate Kinase and DNA*

Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of γ-phosphates from nucleoside triphosphates to nucleoside diphosphates through an invariant histidine residue. It has been reported that the high-energy phosphorylated enzyme intermediate exhibits a protein phosphotransferase activity toward the protein histidine kinases CheA and EnvZ, members of the two-component signal transduction systems in bacteria. Here we demonstrate that the apparent protein phosphotransferase activity of NDP kinase occurs only in the presence of ADP, which can mediate the phosphotransfer from the phospho-NDP kinase to the target enzymes in catalytic amounts (∼1 nm). These findings suggest that the protein kinase activity of NDP kinase is probably an artifact attributable to trace amounts of contaminating ADP. Additionally, we show thatEscherichia coli NDP kinase, like its human homologue NM23-H2/PuF/NDP kinase B, can bind and cleave DNA. Previous in vivo functions of E. coli NDP kinase in the regulation of gene expression that have been attributed to a protein phosphotransferase activity can be explained in the context of NDP kinase-DNA interactions. The conservation of the DNA binding and DNA cleavage activities between human and bacterial NDP kinases argues strongly for the hypothesis that these activities play an essential role in NDP kinase function in vivo.

Escherichia coli nucleoside diphosphate kinase is a uracil-processing DNA repair nuclease.

Escherichia coli nucleoside diphosphate kinase (eNDK) is an XTP:XDP phosphotransferase that plays an important role in the regulation of cellular nucleoside triphosphate concentrations. It is also one of several recently discovered DNases belonging to the NM23/NDK family. E. coli cells disrupted in the ndk gene display a spontaneous mutator phenotype, which has been attributed to the mutagenic effects of imbalanced nucleotide pools and errors made by replicative DNA polymerases. Another explanation for the increased mutation rates is that endk- cells lack the nuclease activity of the NDK protein that is essential for a DNA repair pathway. Here, we show that purified, cloned endk is a DNA repair nuclease whose substrate is uracil misincorporated into DNA. We have identified three new catalytic activities in eNDK that act sequentially to repair the uracil lesion: (i) uracil-DNA glycosylase that excises uracil from single-stranded and from U/A and U/G mispairs in double-stranded DNA; (ii) apyrimidinic endonuclease that cleaves double-stranded DNA as a lyase by forming a covalent enzyme-DNA intermediate complex with the apyrimidinic site created by the glycosylase; and (iii) DNA repair phosphodiesterase that removes 3′-blocking residues from the ends of duplex DNA. All three of these activities, as well as the nucleoside-diphosphate kinase, reside in the same protein. Based on these findings, we propose an editing function for eNDK as a mechanism by which the enzyme prevents mutations in DNA.

The requirement of simian virus 40 gene A product for the stimulation of cellular thymidine kinase activity after viral infection.

Abstract Mouse cells in culture contain two distinct forms of thymidine kinase enzyme activities. These two enzymes have been separated by polyacrylamide gel electrophoresis into a 0.2 R f and a 0.5 R f activity. The 0.2 R f enzyme was found only in actively growing cells, while the 0.5 R f form of thymidine kinase is the mitochondrial-associated enzyme and is most prominent in resting cells in culture. SV40 infection of these resting cells results in an increased specific activity of only the 0.2 R f form of this enzyme. SV40 wild-type and SV40 ts BC and ts C mutants stimulated the levels of the 0.2 R f thymidine kinase in resting cells after viral infection at either the permissive temperature (32°) or the nonpermissive temperature (41°). Five different SV40 ts A mutants ( ts A7, 28, 30, 58, and 209) and two different SV40 ts D mutants ( ts D202, 270) only stimulated thymidine kinase activity at the permissive temperature. Little or no 0.2 R f thymidine kinase activity could be detected in ts A or ts D mutant-infected cells at the nonpermissive temperature. The SV40 tsA255 mutant appeared to be an exception to the A mutant class in that it stimulated the 0.2 R f thymidine kinase activity at both permissive and nonpermissive temperatures. These results indicate that the SV40 A gene product may be required directly, and the D gene product indirectly, in the stimulation of cellular enzyme activities following viral infection.

Modulation of c‐myc Transcription by Triple Helix Formationa

The human c-myc oncogene promoter was used as a model with which to study the mechanism of action of oligodeoxyribonucleotides targeted to a gene regulatory region. The nuclease-hypersensitive element, NHE, lying -115 bp from the P1 promoter of the human c-myc gene, is known to be required in cis for transcription of the gene from both P1 and P2 promoters (Fig. 1). Inhibition of c-myc transcription by an oligonucleotide designed to bind to NHE by triplex formation has been observed in a cell-free transcription assay. Using a reconstituted transcription system with the semipurified PuF transcription factor whose site of interaction resides within the NHE, it is shown here that the oligonucleotide inhibits PuF-mediated transcription. These findings, together with data presented elsewhere showing that: (1) PU1 binds to cloned DNA fragments to form a colinear triplex; (2) PU1 inhibits transcription in nuclear extracts; (3) triple helix formation inhibits the binding of PuF to its target NHE element in an in vitro binding competition assay (E. Postel, R. Durland, and M. Hogan, submitted); (4) triplex formation at the NHE target site can occur in living HeLa cells treated with the triplex-forming PU1 oligomer, and (5) c-myc mRNA synthesis in these treated cells is repressed, clearly support the proposed model in which the oligonucleotide targeted against the c-myc NHE promoter region binds to form a triplex, thereby blocking access to the regulatory protein PuF. This results in promoter-sensitive repression of transcriptional activation of the c-myc gene. The potential for manipulation of gene expression by oligonucleotides targeted to a DNA sequence of the c-myc oncogene promoter and other gene promoters is clear.

In vivo and in vitro phosphorylation of the adenovirus type 5 single-strand-specific DNA-binding protein

Abstract The type 5 adenovirus single-strand-specific DNA-binding protein can be labeled with 32 PO 4 added as inorganic phosphate to the medium of infected monkey or human cells. The DNA binding protein in the infected cell can be isolated as a phosphoprotein at early and late times after infection. Most or all of the [ 32 P]phosphate can be removed from the DNA-binding protein by alkaline phosphatase or hydrolysis at alkaline pH. The dephosphorylated protein retains the ability to bind specifically to single-stranded DNA. H5 ts 125 is a mutation in the structural gene of the 72,000 MW single-strand-specific DNA-binding protein. When cells infected with this viral mutant at the permissive temperature are shifted to the nonpermissive temperature, the phosphorylation of the 72,000 MW protein decreased rapidly (within 30 min) even though approximately normal (wild type) levels of this protein (labeled with 35 S]methionine for 30 min) were detected in H5 ts 125-infected cells shifted to the nonpermissive temperature. An in vitro protein kinase assay utilizing nuclei from infected or uninfected cells has been developed. This in vitro assay can detect the phosphorylation of at least three adenovirus-induced phosphoproteins (100,000 MW; 72,000 MW; 36,000 MW) that are also observed when adenovirus-infected cells are labeled with inorganic [ 32 P] phosphate in vivo . The protein kinase activity detected in this in vitro assay is not dependent upon or stimulated by the addition of exogenous cyclic AMP or cyclic GMP. The 72,000 M W protein phosphorylated in vitro by this phosphokinase activity has been shown to be the adenovirus single-strand-specific DNA-binding protein by a specific immunoprecipitation test.