Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sequence of Escherichia coli UvrA protein suggests that it may fold into two functional domains each possessing DNA binding and ATPase activities. We have taken two approaches to physically isolate polypeptides corresponding to the two putative domains. First, a 180 base pair DNA segment encoding multiple collagenase recognition sequences was inserted into UvrA's putative interdomain hinge region. This UvrA derivative was purified and digested with collagenase, and the resulting 70-kDa N-terminal and 35-kDa C-terminal fragments were purified. Both fragments possessed nonspecific DNA binding activity, but only the N-terminal domain retained its nucleotide binding capacity as evidence by measurements of ATP hydrolysis and by ATP photo-cross-linking. Together, the two fragments failed to substitute for UvrA in reconstituting (A)BC excinuclease and, therefore, were presumed to be unable to load UvrB onto damaged DNA. Second, the DNA segments encoding the two domains were fused to the beta-galactosidase gene. The UvrA N-terminal domain-beta-galactosidase fusion protein was overproduced and purified. This fusion protein had ATPase activity, thus confirming that the amino-terminal domain does possess an intrinsic ATPase activity independent of any interaction with the carboxy terminus. Our results show that UvrA has two functional domains and that the specificity for binding to damaged DNA is provided by the proper three-dimensional orientation of one zinc finger motif relative to the other and is not an intrinsic property of an individual zinc finger domain.
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PMID:Isolation and characterization of functional domains of UvrA. 182 51

The Escherichia coli UvrA protein possesses a stretch of amino acids, 494 to 513, that matches the consensus sequence of the helix-turn-helix motif of many sequence-specific DNA binding proteins. It also has two zinc finger motif regions and two ATP binding sites. To study the potential roles of both helix-turn-helix and zinc finger motifs in the functioning of UvrA protein, random mutations were created in these motif regions by degenerate oligonucleotide-directed mutagenesis. Using this method, 12 single substitution mutants (eight in the helix-turn-helix motif region, one in the N-terminal zinc finger region, and three in the C-terminal zinc finger region) were isolated that failed to confer UV resistance in the E. coli strain deleted of the uvrA gene. One "hyper" UV-resistant mutant, G275A, was identified that conferred significantly more UV resistance than the wild type in the MH1-delta A strain. To further investigate the mechanism of failure of these mutant UvrA proteins to support nucleotide excision repair, two mutant UvrA proteins, G502D and V508D, were selected for purification and characterization, since they carry mutations at the positions offered as the putative constellation for the helix-turn-helix motif. The binding affinity of these two mutants for nonirradiated plasmid DNA was unaffected by the mutations. Both mutant proteins exhibited substantial ATPase activity, and together with the UvrB protein, they were capable of generating positively supercoiled plasmid DNA from the relaxed form in the presence of ATP and bacterial topoisomerase I. However, both mutant proteins failed to respond to UV damage in the filter binding assay and were incapable of forming 2 x SSC-resistant nucleoprotein complexes with UvrB protein on UV-irradiated plasmid DNA. Taking these properties together, it appears that the mutations in the helix-turn-helix motif region impaired the UvrA protein's ability to recognize UV damage, while its other activities were largely unaffected. Interestingly, ERCC-3, a human DNA repair protein, also has a similar helix-turn-helix motif. Given the highly conserved nature of repair proteins in general, this observation raises the possibility that both procaryotes and eucaryotes might use similar mechanisms to recognize damaged sites in their genomes.
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PMID:Mutations in the helix-turn-helix motif of the Escherichia coli UvrA protein eliminate its specificity for UV-damaged DNA. 844 6

Hydrolysis of guanosine triphosphate (GTP) by the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor-1 (ARF1) depends on a GTPase-activating protein (GAP). A complementary DNA encoding the ARF1 GAP was cloned from rat liver and predicts a protein with a zinc finger motif near the amino terminus. The GAP function required an intact zinc finger and additional amino-terminal residues. The ARF1 GAP was localized to the Golgi complex and was redistributed into a cytosolic pattern when cells were treated with brefeldin A, a drug that prevents ARF1-dependent association of coat proteins with the Golgi. Thus, the GAP is likely to be recruited to the Golgi by an ARF1-dependent mechanism.
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PMID:The ARF1 GTPase-activating protein: zinc finger motif and Golgi complex localization. 853 93

DnaJ is a molecular chaperone, which not only binds to its various protein substrates, but can also activate the DnaK cochaperone to bind to its various protein substrates as well. DnaJ is a modular protein, which contains a putative zinc finger motif of unknown function. Quantitation of the released Zn(II) ions, upon challenge with p-hydroxymercuriphenylsulfonic acid, and by atomic absorption showed that two Zn(II) ions interact with each monomer of DnaJ. Following the release of Zn(II) ions, the free cysteine residues probably form disulfide bridge(s), which contribute to overcoming the destabilizing effect of losing Zn(II). Supporting this view, infrared and circular dichroism studies show that the DnaJ secondary structure is largely unaffected by the release of Zn(II). Moreover, infrared spectra recorded at different temperatures, as well as scanning calorimetry, show that the Zn(II) ions help to stabilize DnaJ's tertiary structure. An internal 57-amino acid deletion of the cysteine-reach region did not noticeably affect the affinity of this mutant protein, DnaJDelta144-200, to bind DnaK nor its ability to stimulate DnaK's ATPase activity. However, the DnaJDelta144-200 was unable to induce DnaK to a conformation required for the stabilization of the DnaK-substrate complex. Additionally, the DnaJDelta144-200 mutant protein alone was unimpaired in its ability to interact with its final sigma32 transcription factor substrate, but exhibited reduced affinity toward its P1 RepA and lambdaP substrates. Finally, these in vitro results correlate well with the in vivo observed partial inhibition of bacteriophage lambda growth in a DnaJDelta144-200 mutant background.
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PMID:Structure-function analysis of the zinc finger region of the DnaJ molecular chaperone. 866 61

We identified, in the facultative intracellular pathogen Listeria monocytogenes, a previously unknown Clp ATPase, unique among the HSP100 proteins because of the presence of a short N-terminal region with a potential zinc finger motif. This protein of 726 amino acids is highly homologous to ClpE of Bacillus subtilis, and is a member of a new subfamily of HSP100/Clp ATPases. The clpE gene is transcribed as a monocistronic mRNA from a typical consensus sigma A promoter. clpE is not stimulated by various stresses, but is upregulated in a clpC mutant. This is the first example of cross-regulation between Clp ATPases. By constructing a clpE mutant of L. monocytogenes, we found that ClpE is required for prolonged survival at 42 degrees C and is involved in the virulence of this pathogen. A double mutant deficient in both ClpE and ClpC was avirulent in a mouse model and completely eliminated in the liver. Electron microscopy studies did not show any morphological alterations in clpE or clpC mutants. In the clpE-clpC double mutant, however, cell division was affected, indicating that ClpE acts synergistically with ClpC in cell septation. These results show that the Clp chaperones play a crucial role in both cell division and virulence of L. monocytogenes.
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PMID:ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes. 998 21

Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex that is composed of the products of the UL5, UL52, and UL8 genes. A subcomplex consisting of the UL5 and UL52 proteins retains all the enzymatic activities exhibited by the holoenzyme in vitro. The UL52 protein contains a putative zinc finger at its C terminus which is highly conserved among both prokaryotic and eukaryotic primases. We constructed a mutation in which two highly conserved cysteine residues in the zinc finger motif were replaced with alanine residues. A UL52 expression plasmid containing the mutation in the zinc finger region is unable to support the growth of a UL52 mutant virus in a transient complementation assay. Wild type and mutant UL5.UL52 subcomplexes were purified from insect cells infected with recombinant baculoviruses. Surprisingly, the mutant protein was severely affected in all biochemical activities tested; no helicase or primase activities could be detected, and the mutant protein retains only about 9% of wild type levels of single-stranded DNA-dependent ATPase activity. Gel mobility shift assays showed that DNA binding is severely affected as well; the mutant subcomplex only retains approximately 8% of wild type levels of binding to a forked substrate. On the other hand, the mutant protein retains its ability to interact with UL5 as indicated by copurification and with UL8 as indicated by a supershifted band in the gel mobility shift assay. In addition, the ability of individual subunits to bind single-stranded DNA was examined by photo cross-linking. In the wild type UL5.UL52 subcomplex, both subunits are able to bind an 18-mer of oligo(dT). The mutant subcomplex was severely compromised in the ability of both UL5 and UL52 to bind the oligonucleotide; total cross-linking was only 2% of wild type levels. These results are consistent with the proposal that the putative zinc binding motif of UL52 is required not only for binding of the UL52 subunit to DNA and for primase activity but also for optimal binding of UL5 to DNA and for the subsequent ATPase and helicase activities.
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PMID:A mutation in the C-terminal putative Zn2+ finger motif of UL52 severely affects the biochemical activities of the HSV-1 helicase-primase subcomplex. 1007 7

To complement evidence for nucleoside triphosphate phosphohydrolase (NTPase), RNA helicase, RNA 5' triphosphate phosphohydrolase, and nucleic acid-binding activities by the core shell protein lambda1 of mammalian orthoreoviruses (reoviruses), we determined nucleotide sequences of the lambda1-encoding L3 gene segments from three isolates: type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D). The T1L and T3D L3 gene sequences and deduced lambda1 protein sequences shared high levels of identity (97.7% and 99.3%, respectively). The lambda1 sequences differed at only 9 of 1275 amino acids. Two single-nucleotide insertions relative to a previously published sequence for T3D L3 extended the lambda1 open reading frame to within 60 nucleotides of the plus-strand 3' end for T3D and the other isolates sequenced, in keeping with the short 3' nontranslated regions of the other nine gene segments. Seven of the nine amino acid differences between T1L and T3D lambda1 were located within the amino-terminal 500 residues of lambda1, a region with putative sequence similarities to NTPases and RNA helicases. The T2J L3 and lambda1 sequences were found to be more divergent, especially within the amino-terminal 180 residues of lambda1, preceding the putative CCHH zinc finger motif. The T2J L3 sequence, along with partial sequences for L3 genes from three other reovirus isolates, suggested that one or more of the polymorphisms at amino acids 71, 215, 500, 1011, and/or 1100 in lambda1 contribute to the L3-determined differences in ATPase activities by T1L and T3D cores. The findings contribute to our ongoing efforts to elucidate sequence-structure-function relationships for the lambda1 core protein.
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PMID:Mammalian reovirus L3 gene sequences and evidence for a distinct amino-terminal region of the lambda1 protein. 1032 67

Very limited information is available on the role of phosphatidylinositol 3-phosphate (PI[3]P) in vesicle trafficking in plant cells. To investigate the role of PI(3)P during the vesicle trafficking in plant cells, we exploited the PI(3)P-specific binding property of the endosome binding domain (EBD) (amino acids 1257 to 1411) of human early endosome antigen 1, which is involved in endosome fusion. When expressed transiently in Arabidopsis protoplasts, a green fluorescent protein (GFP):EBD fusion protein exhibited PI(3)P-dependent localization to various compartments--such as the trans-Golgi network, the prevacuolar compartment, the tonoplasts, and the vesicles in the vacuolar lumen--that varied with time. The internalized GFP:EBD eventually disappeared from the lumen. Deletion experiments revealed that the PI(3)P-dependent localization required the Rab5 binding motif in addition to the zinc finger motif. Overexpression of GFP:EBD inhibited vacuolar trafficking of sporamin but not trafficking of H(+)-ATPase to the plasma membrane. On the basis of these results, we propose that the trafficking of GFP:EBD reflects that of PI(3)P and that PI(3)P synthesized at the trans-Golgi network is transported to the vacuole through the prevacuolar compartment for degradation in plant cells.
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PMID:Trafficking of phosphatidylinositol 3-phosphate from the trans-Golgi network to the lumen of the central vacuole in plant cells. 1122 86

The ClpX heat shock protein of Escherichia coli is a member of the universally conserved Hsp100 family of proteins, and possesses a putative zinc finger motif of the C(4) type. The ClpX is an ATPase which functions both as a substrate specificity component of the ClpXP protease and as a molecular chaperone. Using an improved purification procedure we show that the ClpX protein is a metalloprotein complexed with Zn(II) cations. Contrary to other Hsp100 family members, ClpXZn(II) exists in an oligomeric form even in the absence of ATP. We show that the single ATP-binding site of ClpX is required for a variety of tasks, namely, the stabilization of the ClpXZn(II) oligomeric structure, binding to ClpP, and the ClpXP-dependent proteolysis of the lambdaO replication protein. Release of Zn(II) from ClpX protein affects the ability of ClpX to bind ATP. ClpX, free of Zn(II), cannot oligomerize, bind to ClpP, or participate in ClpXP-dependent proteolysis. We also show that ClpXDeltaCys, a mutant protein whose four cysteine residues at the putative zinc finger motif have been replaced by serine, behaves in similar fashion as wild type ClpX protein whose Zn(II) has been released either by denaturation and renaturation, or chemically by p-hydroxymercuriphenylsulfonic acid.
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PMID:Structure-function analysis of the zinc-binding region of the Clpx molecular chaperone. 1127 49

Mutations in the ATRX gene cause a severe X-linked mental retardation syndrome that is frequently associated with alpha thalassemia (ATR-X syndrome). The previously characterized ATRX protein (approximately 280 kDa) contains both a Plant homeodomain (PHD)-like zinc finger motif as well as an ATPase domain of the SNF2 family. These motifs suggest that ATRX may function as a regulator of gene expression, probably by exerting an effect on chromatin structure, although the exact cellular role of ATRX has not yet been fully elucidated. Here we characterize a truncated (approximately 200 kDa) isoform of ATRX (called here ATRXt) that has been highly conserved between mouse and human. In both species, ATRXt arises due to the failure to splice intron 11 from the primary transcript, and the use of a proximal intronic poly(A) signal. We show that the relative expression of the full length and ATRXt isoforms is subject to tissue-specific regulation. The ATRXt isoform contains the PHD-like domain but not the SWI/SNF-like motifs and is therefore unlikely to be functionally equivalent to the full length protein. We used indirect immunofluorescence to demonstrate that the full length and ATRXt isoforms are colocalized at blocks of pericentromeric heterochromatin but unlike full length ATRX, the truncated isoform does not associate with promyelocytic leukemia (PML) nuclear bodies. The high degree of conservation of ATRXt and the tight regulation of its expression relative to the full length protein suggest that this truncated isoform fulfills an important biological function.
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PMID:A conserved truncated isoform of the ATR-X syndrome protein lacking the SWI/SNF-homology domain. 1472 60


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