Biomedical subjects

Edith H Postel

Publications and source records attributed to Edith H Postel.

6 recordsLinked to original sources

PubMed2006

Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung.

Escherichia coli nucleoside-diphosphate kinase (Ndk) catalyzes nucleoside triphosphate synthesis and maintains intracellular triphosphate pools. Mutants of E. coli lacking Ndk exhibit normal growth rates but show a mutator phenotype that cannot be entirely attributed to the absence of Ndk catalytic activity or to an imbalance in cellular triphosphates. It has been suggested previously that Ndk, similar to its human counterparts, possesses nuclease and DNA repair activities, including the excision of uracil from DNA, an activity normally associated with the Ung and Mug uracil-DNA glycosylases (UDGs) in E. coli. Here we have demonstrated that recombinant Ndk purified from wild-type E. coli contains significant UDG activity that is not intrinsic, but rather, is a consequence of a direct physical and functional interaction between Ung and Ndk, although a residual amount of intrinsic UDG activity exists as well. Co-purification of Ung and Ndk through multicolumn low pressure and nickel-nitrilotriacetic acid affinity chromatography suggests that the interaction occurs in a cellular context, as was also suggested by co-immunoprecipitation of endogenous Ung and Ndk from cellular extracts. Glutathione S-transferase pulldown and far Western analyses demonstrate that the interaction also occurs at the level of purified protein, suggesting that it is specific and direct. Moreover, significant augmentation of Ung catalytic activity by Ndk was observed, suggesting that the interaction between the two enzymes is functionally relevant. These findings represent the first example of Ung interacting with another E. coli protein and also lend support to the recently discovered role of nucleoside-diphosphate kinases as regulatory components of multiprotein complexes.

Blotting, Far-Western↗

PubMed2003

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.

Chromatography↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2002

Structure-based mutational and functional analysis identify human NM23-H2 as a multifunctional enzyme.

The human NM23-H2 protein is a transcriptional regulator (PuF) that binds and cleaves DNA via covalent bond formation, and also catalyzes phosphoryl transfer (NDP kinase). Our previous work has identified two separate DNA-binding regions on NM23-H2/PuF: a sequence-dependent DNA-binding surface involving residues Arg34, Asn69, and Lys134 on the equator of the hexameric protein and a covalent DNA-binding site involving Lys12 located in the nucleotide-binding site, the site of the NDP kinase reaction. To understand the role of the nucleotide-binding site in the DNA cleavage reaction and to establish a connection between the nuclease and the NDP kinase activities, we used the known crystal structure of NM23-H2 complexed with GDP as the basis for site-directed mutagenesis. We thus identified Arg88 and Arg105 as residues that are, in addition to Lys12, critical for covalent DNA binding and DNA cleavage, as well as for the NDP kinase reaction. Another residue, Gln17, was required only for DNA cleavage, and Tyr52, Asn115, and His118 were found to be essential only for the NDP kinase activity. Six of these seven functionally important amino acids associated with the nucleotide-binding site are evolutionarily conserved, underscoring their biological importance. We also show that nucleoside triphosphates but not nucleoside diphosphates inhibited the covalent DNA binding and DNA cleavage reactions, independent of phosphoryl transfer and the NDP kinase reaction. These findings collectively suggest that the binding modes of mononucleotides and duplex DNA oligonucleotides in the nucleotide-binding site differ, and that NM23-H2 possesses multiple biochemical activities. A model consistent with these observations is presented.

Adenosine Diphosphate↗

PubMed2001

Interactions between Escherichia coli nucleoside-diphosphate kinase and DNA.

Nucleoside-diphosphate (NDP) kinase (NTP:nucleoside-diphosphate phosphotransferase) catalyzes the reversible transfer of gamma-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 (approximately 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 that Escherichia 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.

Adenosine Diphosphate↗

PubMed2001

NM23-H1 and NM23-H2 repress transcriptional activities of nuclease-hypersensitive elements in the platelet-derived growth factor-A promoter.

The platelet-derived growth factor (PDGF)-A promoter is regulated by a number of GC-rich regulatory elements that possess non-B-form DNA structures. Screening of a HeLa cDNA expression library with the C-rich strand of a PDGF-A silencer sequence (5'-S1 nuclease-hypersensitive site (SHS)) yielded three cDNA clones encoding NM23-H1, a protein implicated as a suppressor of metastasis in melanoma and breast carcinoma. Recombinant human NM23-H1 cleaved within the 3'-portions of both 5'-SHS strands in either single-stranded or duplex forms. In contrast, NM23-H2, known as a transcriptional activator with a DNA cleavage function, cleaved within the 5'-portions of both strands, revealing that NM23-H1 and NM23-H2 cleave at distinct sites of the 5'-SHS and by different mechanisms. NM23-H1 and NM23-H2 also cleaved within the PDGF-A basal promoter region, again exhibiting preferences for cleavage within the 5'- and 3'-portions of the element, respectively. Transient transfection analyses in HepG2 cells revealed that both NM23-H1 and -H2 repressed transcriptional activity driven by the PDGF-A basal promoter (-82 to +8). Activity of the negative regulatory region (-1853 to -883), which contains the 5'-SHS, was also inhibited modestly by NM23-H1 and NM23-H2. These studies demonstrate for the first time that NM23-H1 interacts both structurally and functionally with DNA. They also indicate a role for NM23 proteins in repressing transcription of a growth factor oncogene, providing a possible molecular mechanism to explain their metastasis-suppressing effects.

Antigens, Neoplasm↗