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1.
J Biochem Mol Biol ; 40(1): 7-14, 2007 Jan 31.
Article in English | MEDLINE | ID: mdl-17244476

ABSTRACT

Helicases are ubiquitous enzymes, which utilize the energy liberated during nucleotide triphosphate hydrolysis to separate double-stranded nucleic acids into single strands. These enzymes are very attractive targets for the development of new antibacterial compounds. The PcrA DNA helicase from Staphylococcus aureus is a good candidate for drug discovery. This enzyme is unique in the genome of S. aureus and essential for this bacterium. Furthermore, it has recently been published that it is possible to identify inhibitors of DNA helicases such as PcrA. In this report, we study the properties of recombinant PcrA from S. aureus purified from Escherichia coli to develop ATPase and helicase assays to screen for inhibitors.


Subject(s)
Bacterial Proteins/analysis , DNA Helicases/analysis , Drug Evaluation, Preclinical/methods , Recombinant Proteins/analysis , Staphylococcus aureus/enzymology , Adenosine Triphosphatases/metabolism , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cloning, Molecular , DNA Helicases/antagonists & inhibitors , DNA Helicases/genetics , DNA Helicases/metabolism , Recombinant Proteins/metabolism , Research Design
2.
Oncol Res ; 16(12): 549-56, 2007.
Article in English | MEDLINE | ID: mdl-18351129

ABSTRACT

Helicases are present in viruses, prokaryotes, and eukaryotes, and several of them have been linked to human diseases. Here we study the role of one putative DEAD-box RNA helicase, MrDb (DDX18), in tumor cells. We show that MrDb is a nucleolar protein ubiquitously expressed in tumor cells and that it is more abundantly expressed in proliferating cells. Inhibition of MrDb with dominant negative mutant or a shRNA reduces tumor cell proliferation without inducing a cell cycle arrest or apoptosis. These findings suggest that MrDb is important for cell proliferation and that its inhibition could prevent tumor cell proliferation.


Subject(s)
Cell Proliferation , DEAD-box RNA Helicases/metabolism , Gene Expression/physiology , Apoptosis/physiology , Cell Cycle/physiology , Cell Line, Tumor , Drug Delivery Systems , Humans , Mutation , RNA/metabolism
3.
Biochem Biophys Res Commun ; 341(3): 828-36, 2006 Mar 17.
Article in English | MEDLINE | ID: mdl-16442499

ABSTRACT

Helicases contain conserved motifs involved in ATP/magnesium/nucleic acid binding and in the mechanisms coupling nucleotide hydrolysis to duplex unwinding. None of these motifs are located at the adenine-binding pocket of the protein. We show here that the superfamily I helicase, helicase IV from Escherichia coli, utilizes a conserved glutamine and conserved aromatic residue to interact with ATP. Other superfamily I helicases such as, UvrD/Rep/PcrA also possess these residues but in addition they interact with adenine via a conserved arginine, which is replaced by a serine in helicase IV. Mutation of this serine residue in helicase IV into histidine or methionine leads to proteins with unaffected ATPase and DNA-binding activities but with low helicase activity. This suggests that residues located at the adenine-binding pocket, in addition to be involved in ATP-binding, are important for efficient coupling between ATP hydrolysis and DNA unwinding.


Subject(s)
Adenosine Triphosphate/metabolism , DNA Helicases/chemistry , DNA Helicases/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Adenine/chemistry , Adenine/metabolism , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Binding Sites , Conserved Sequence , DNA Helicases/genetics , DNA, Bacterial/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Protein Binding , Protein Structure, Tertiary , Sequence Alignment
4.
Bioorg Med Chem Lett ; 16(4): 923-7, 2006 Feb 15.
Article in English | MEDLINE | ID: mdl-16300943

ABSTRACT

Helicases form an attractive protein family for drug discovery because they are involved in various human diseases. In this report, we show that it is possible to inhibit both the ATPase and the helicase activities of a DNA helicase with dibenzothiepins that bind at its nucleic acid binding site. These results suggest a drug discovery strategy to inhibit DNA helicases.


Subject(s)
DNA Helicases/antagonists & inhibitors , DNA/drug effects , Dibenzothiepins/pharmacology , Enzyme Inhibitors/pharmacology , Escherichia coli Proteins/antagonists & inhibitors , Adenosine Triphosphatases/antagonists & inhibitors , Binding Sites , Binding, Competitive/drug effects , Crystallography, X-Ray , DNA/chemistry , Dibenzothiepins/chemistry , Enzyme Inhibitors/chemistry , Escherichia coli/enzymology , Models, Molecular , Molecular Structure , Protein Conformation , Protein Structure, Tertiary , Structure-Activity Relationship
5.
Nat Struct Mol Biol ; 11(7): 616-22, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15195146

ABSTRACT

The human general transcription factor TFIIH is involved in both transcription and DNA repair. We have identified a structural domain in the core subunit of TFIIH, p62, which is absolutely required for DNA repair activity through the nucleotide excision repair pathway. Using coimmunoprecipitation experiments, we showed that this activity involves the interaction between the N-terminal domain of p62 and the 3' endonuclease XPG, a major component of the nucleotide excision repair machinery. Furthermore, we reconstituted a functional TFIIH particle with a mutant of p62 lacking the N-terminal domain, showing that this domain is not required for assembly of the TFIIH complex and basal transcription. We solved its three-dimensional structure and found an unpredicted pleckstrin homology and phosphotyrosine binding (PH/PTB) domain, uncovering a new class of activity for this fold.


Subject(s)
DNA Repair , Transcription Factors, TFII/physiology , Amino Acid Sequence , DNA-Binding Proteins/metabolism , Endonucleases , Molecular Sequence Data , Nuclear Proteins , Precipitin Tests , Protein Conformation , Sequence Homology, Amino Acid , Transcription Factor TFIIH , Transcription Factors , Transcription Factors, TFII/chemistry
6.
Cell ; 116(4): 541-50, 2004 Feb 20.
Article in English | MEDLINE | ID: mdl-14980221

ABSTRACT

The Rift Valley fever virus (RVFV) is the causative agent of fatal hemorrhagic fever in humans and acute hepatitis in ruminants. We found that infection by RVFV leads to a rapid and drastic suppression of host cellular RNA synthesis that parallels a decrease of the TFIIH transcription factor cellular concentration. Using yeast two hybrid system, recombinant technology, and confocal microscopy, we further demonstrated that the nonstructural viral NSs protein interacts with the p44 component of TFIIH to form nuclear filamentous structures that also contain XPB subunit of TFIIH. By competing with XPD, the natural partner of p44 within TFIIH, and sequestering p44 and XPB subunits, NSs prevents the assembly of TFIIH subunits, thus destabilizing the normal host cell life. These observations shed light on the mechanism utilized by RVFV to evade the host response.


Subject(s)
Cell Nucleus/virology , Rift Valley fever virus/metabolism , Transcription Factors, TFII/chemistry , Animals , Cell Nucleus/metabolism , DNA, Complementary/metabolism , Gene Library , HeLa Cells , Humans , Luciferases/metabolism , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Plasmids/metabolism , Protein Binding , RNA/metabolism , Time Factors , Transcription Factor TFIIH , Transcription Factors, TFII/metabolism , Transcription, Genetic , Two-Hybrid System Techniques
7.
Mol Cell ; 11(6): 1635-46, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12820975

ABSTRACT

Mutations in the XPD gene result in xeroderma pigmentosum (XP) and trichothiodystrophy (TTD), the phenotypes of which are often intricate. To understand the genotype/phenotype relationship, we engineered recombinant TFIIHs in which XPD subunits carry amino acid changes found in XPD patients. We demonstrate that all the XPD mutations are detrimental for XPD helicase activity, thus explaining the NER defect. We also show that TFIIH from TTD patients, but not from XP patients, exhibits a significant in vitro basal transcription defect in addition to a reduced intracellular concentration. Moreover, when XPD mutations prevent interaction with the p44 subunit of TFIIH, transactivation directed by certain nuclear receptors is inhibited, regardless of TTD versus XP phenotype, thus explaining the overlapping symptoms. The implications of these mutations are discussed using a structural model of the XPD protein. Our study provides explanations for the nature and the severity of the various clinical features.


Subject(s)
DNA-Binding Proteins , Hair Diseases/genetics , Proteins/genetics , Transcription, Genetic , Xeroderma Pigmentosum/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Cells, Cultured , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Repair/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , HeLa Cells , Heterozygote , Humans , Insecta , Models, Molecular , Point Mutation , Recombinant Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation , Xeroderma Pigmentosum Group D Protein
8.
Mol Cell ; 10(6): 1391-401, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12504014

ABSTRACT

To counteract the deleterious effects of genotoxic injury, cells have set up a sophisticated network of DNA repair pathways. We show that Gal4-VP16 and RAR transcriptional activators stimulate nucleotide excision repair (NER). This DNA repair activation is not coupled to transcription since it occurs in Cockayne syndrome cells (which are transcription-coupled repair deficient) and is observed in vitro in the presence of alpha-amanitin and in the absence of the basal transcription factors. Using a reconstituted dual incision assay, we also show that binding of activators to their cognate sequences induces a local chromatin remodeling mediated by ATP-driven chromatin remodeling and acetyltransferase activities to facilitate DNA repair.


Subject(s)
DNA Repair/physiology , DNA/metabolism , Trans-Activators/metabolism , Transcription, Genetic , Base Sequence , Binding Sites , Cell Line, Transformed , DNA/chemistry , DNA-Binding Proteins , Herpes Simplex Virus Protein Vmw65/metabolism , Humans , Oligodeoxyribonucleotides/chemistry , Receptors, Retinoic Acid/metabolism , Recombinant Fusion Proteins/metabolism , Restriction Mapping , Saccharomyces cerevisiae Proteins/metabolism , Simian virus 40/genetics , Templates, Genetic , Transcription Factors/metabolism , Transfection
9.
J Biol Chem ; 277(35): 31761-7, 2002 Aug 30.
Article in English | MEDLINE | ID: mdl-12080057

ABSTRACT

To further our understanding of the transcription/DNA repair factor TFIIH, we investigated the role of its p52 subunit in TFIIH function. Using a completely reconstituted in vitro transcription or nucleotide excision repair (NER) system, we show that deletion of the C-terminal region of p52 results in a dramatic reduction of TFIIH NER and transcription activities. This mutation prevents promoter opening and has no effect on the other enzymatic activities of TFIIH. Moreover, we demonstrate that intact p52 is needed to anchor the XPB helicase within TFIIH, providing an explanation for the transcription and NER defects observed with the mutant p52. We show that these two subunits physically interact and map domains involved in the interface. Taken together, our results show that the p52/Tfb2 subunit of TFIIH regulates the function of XPB through pair-wise interactions as described previously for p44 and XPD.


Subject(s)
DNA Repair , DNA-Binding Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors, TFII , Transcription Factors/genetics , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing , Base Sequence , Binding Sites , DNA Helicases , DNA-Binding Proteins/genetics , Humans , Mutagenesis , Promoter Regions, Genetic , Recombinant Proteins/metabolism , Sequence Deletion , Transcription Factor TFIIH , Transcription, Genetic
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