RESUMO
In most cases, xeroderma pigmentosum group D (XP-D) and trichothiodystrophy (TTD) patients carry mutations in the carboxy-terminal domain of the evolutionarily conserved helicase XPD, which is one of the subunits of the transcription/repair factor TFIIH (refs 1,2). In this study, we demonstrate that XPD interacts specifically with p44, another subunit of TFIIH, and that this interaction results in the stimulation of 5'-->3' helicase activity. Mutations in the XPD C-terminal domain, as found in most patients, prevent the interaction with p44, thus explaining the decrease in XPD helicase activity and the nucleotide excision repair (NER) defect.
Assuntos
DNA Helicases/genética , Reparo do DNA , Proteínas de Ligação a DNA , Doenças do Cabelo/genética , Proteínas/genética , Fatores de Transcrição TFII , Fatores de Transcrição/metabolismo , Xeroderma Pigmentoso/genética , DNA Helicases/metabolismo , Humanos , Mutação , Conformação Proteica , Proteínas/metabolismo , Fator de Transcrição TFIIH , Proteína Grupo D do Xeroderma PigmentosoRESUMO
Using the human basal transcription factors TFIID and TFIIH as examples, we show that pairwise coexpression of polypeptides in Escherichia coli can be used as a tool for the identification of specifically interacting subunits within multiprotein complexes. We find that coexpression of appropriate combinations generally leads to an increase in the solubility and stability of the polypeptides involved, which means that large amounts of the resulting complexes can immediately be obtained for subsequent biochemical and structural analysis. Furthermore, we demonstrate that the solubilization and/or the proper folding of a protein by its natural partner can be used as a monitor for deletion mapping to determine precise interaction domains. Coexpression can be used as an alternative or complementary approach to conventional techniques for interaction studies such as yeast two-hybrid analysis, GST pulldown and immunoprecipitation.
Assuntos
Escherichia coli/genética , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sítios de Ligação , Expressão Gênica , Vetores Genéticos/genética , Humanos , Substâncias Macromoleculares , Modelos Moleculares , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Solubilidade , Fator de Transcrição TFIID , Fator de Transcrição TFIIH , Fatores de Transcrição/genética , Fatores de Transcrição TFII/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Poly-ADP ribosylation of nuclear proteins is activated when poly(ADP-ribose) polymerase (PARP), a nuclear zinc-finger enzyme, binds to single-strand DNA breaks. To understand how the signal emerging from its DNA-binding domain (DBD) bound to such breaks is transduced to its catalytic domain, the structure-function relationship of the DBD was investigated. We have used mutagenesis by the polymerase chain reaction (PCR) to generate a random library of PARP mutants. In this work, we describe the identification of catalytically inactive mutants bearing single point mutations, located outside the two zinc fingers in the DBD, that have conserved their full capacity to bind DNA. The results obtained demonstrate that the DNA-dependent activation of PARP requires not only a capacity to bind DNA but also a number of crucial residues to maintain a conformation of the domain necessary to transfer an 'activation signal' to the catalytic domain.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Catálise , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Humanos , Mutagênese , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Dissection of the human poly(ADP-ribose) polymerase (PARP) molecule in terms of its structure-function relationship has proved to be an essential step towards understanding the biological role of poly(ADP-ribosylation) as a cellular response to DNA damage in eukaryotes. Current approaches aimed at elucidating the implication of this multifunctional enzyme in the maintenance of the genomic integrity will be presented.
Assuntos
Reparo do DNA , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/fisiologia , Animais , Catálise , Galinhas , Cristalização , Cristalografia por Raios X , Dano ao DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Genoma Humano , Células HeLa , Humanos , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Relação Estrutura-Atividade , TransfecçãoAssuntos
Participação do Paciente , Encaminhamento e Consulta , Tomada de Decisões , Feminino , HumanosAssuntos
Complicações do Trabalho de Parto , Cordão Umbilical , Feminino , Humanos , Gravidez , ProlapsoAssuntos
Estrogênios Conjugados (USP)/farmacologia , Menopausa/efeitos dos fármacos , Noretindrona/farmacologia , Amenorreia/etiologia , Atrofia , Endométrio/patologia , Estrogênios Conjugados (USP)/administração & dosagem , Feminino , Humanos , Lipídeos/sangue , Noretindrona/administração & dosagem , Tromboflebite/induzido quimicamenteAssuntos
Irrigação Terapêutica , Inércia Uterina/terapia , Adulto , Europa (Continente) , Feminino , Grécia , História do Século XIX , História do Século XX , História Antiga , Humanos , Métodos , Obstetrícia/história , Hemorragia Pós-Parto/etiologia , Hemorragia Pós-Parto/prevenção & controle , Gravidez , Cloreto de Sódio/administração & dosagem , Temperatura , Estados Unidos , Inércia Uterina/complicaçõesAssuntos
Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônios/uso terapêutico , Leiomioma/cirurgia , Neoplasias Uterinas/cirurgia , Ensaios Clínicos como Assunto , Terapia Combinada , Preparações de Ação Retardada , Feminino , Hormônio Liberador de Gonadotropina/uso terapêutico , Humanos , Leiomioma/tratamento farmacológico , Leuprolida , Distribuição Aleatória , Neoplasias Uterinas/tratamento farmacológicoRESUMO
PIP: Drs. Edelman and Bergers' report "Contraceptive practice and tuboovarian abscess (Am. J. Obstet. Gynecol. 138:541, 1980) may produce the impression in the medical profession that the IUD does not predispose to salpingitis, salpingo-oophoritis, and tubo-ovarian abscess, as published data and clinical experience would suggest. Also, the diagnostic criteria for diagnosing 'acute pelvic inflammatory disease' stated in the report, and published studies of Jacobson and Westrom and Chaparro et al question a diagnosis of pelvic inflammatory disease that is not confirmed endoscopically or by some direct visualization obtained surgically. 35% of patients who had laparoscopy by Jacobson and Westrom and who were suspected of having salpingitis, or pelvic inflammatory disease, and 54% of laparoscoped patients suspected by Chaparoo et.al. of having pelvic inflammatory disease were found not to have either salpingitis or pelvic inflammatory disease of gynecologic etiology. As pelvic inflammation may be caused by a variety of disorders, such as appendicitis, colitis, diverticulitis, and others, the term pelvic inflammatory disease is an imprecise diagnostic term. Edelman and Berger's results can also be questioned on the ground that numerous reports (e.g., Second Report on Intrauterine Contraceptive Devices, Food and Drug Administration, 1978; Population Reports, Series B, No. 3, May 1979, the Johns Hopkins University) indicate an increased incidence of salpingitis with its attendant pelvic crippling pain and infertility that is many times more common in IUD users than in nonIUD users. Available published data therefore strongly suggest that an IUD user is at far greater risk of developing inflammatory disease of infectious etiology in her reproductive tract with its attendant pain, morbidity, infertility, and even death than nonIUD users.^ieng
Assuntos
Dispositivos Intrauterinos/efeitos adversos , Doença Inflamatória Pélvica/etiologia , Feminino , Humanos , GravidezRESUMO
PIP: Letter emphasizing the importance of sex education for unmarried teenagers, stressing findings that the risk of pregnancy soon after beginning intercourse may be greater than that reported in an earlier article, and that unmarried teenagers may be more sexually active than married couples. The writer also notes the importance of including males in sex education efforts, and that education of adults about the need for sex education for children seems called for.^ieng
Assuntos
Adolescente , Educação Sexual , HumanosRESUMO
TAP-p15 heterodimers have been implicated in the export of mRNAs through nuclear pore complexes (NPCs). We report a structural analysis of the interaction domains of TAP and p15 in a ternary complex with a Phe-Gly (FG) repeat of an NPC component. The TAP-p15 heterodimer is structurally similar to the homodimeric transport factor NTF2, but unlike NTF2, it is incompatible with either homodimerization or Ran binding. The NTF2-like heterodimer functions as a single structural unit in recognizing an FG repeat at a hydrophobic pocket present only on TAP and not on p15. This FG binding site interacts synergistically with a second site at the C terminus of TAP to mediate mRNA transport through the pore. In general, our findings suggest that FG repeats bind with a similar conformation to different classes of transport factors.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transporte Ativo do Núcleo Celular/fisiologia , Proteínas de Transporte/química , Poro Nuclear/química , Proteínas Nucleares/química , Proteínas de Transporte Nucleocitoplasmático , RNA Mensageiro/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Poro Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Proteína ran de Ligação ao GTP/metabolismoRESUMO
TFIIH is a multiprotein complex required for both transcription and DNA repair. Single particles of human TFIIH were revealed by electron microscopy and image processing at a resolution of 3.8 nm. TFIIH is 16 x 12.5 x 7.5 nm in size and is organized into a ring-like structure from which a large protein domain protrudes out. A subcomplex assembled from five recombinant core subunits also forms a circular architecture that can be superimposed on the ring found in human TFIIH. Immunolabeling experiments localize several subunits: p44, within the ring structure, forms the base of the protruding protein density which includes the cdk7 kinase, cyclin H, and MAT1. Within the ring structure, p44 was flanked on either side by the XPB and XPD helicases. These observations provide us with a quartenary organizational model of TFIIH.