RESUMO
Topoisomerase IIbeta-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase epsilon. In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G(1) phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Dano ao DNA , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA , Proteínas de Drosophila , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Transglutaminases , Animais , Proteína BRCA1/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Citoplasma/metabolismo , DNA Polimerase II/metabolismo , DNA Complementar/metabolismo , Drosophila , Ecdisona/metabolismo , Proteínas Fúngicas/química , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Camundongos , Proteínas Nucleares , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Fase S , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Transdução de Sinais , Fatores de Tempo , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-HíbridoAssuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , DNA Polimerase Dirigida por DNA/classificação , Humanos , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Pyrococcus furiosus/enzimologia , Pyrococcus furiosus/genética , Homologia de Sequência de AminoácidosRESUMO
Human DNA polymerase epsilon is composed of a 261 kDa catalytic polypeptide and a 55 kDa small subunit of unknown function. cDNAs encoding the small subunit of human and mouse DNA polymerase epsilon were cloned. The predicted polypeptides have molecular masses of 59.469 and 59.319 kDa respectively and they are 90% identical. The human and mouse polypeptides show 22% identity with the 80 kDa subunit of the five subunit DNA polymerase epsilon from the yeast Saccharomyces cerevisiae. The high degree of conservation suggests that the 55 kDa subunit shares an essential function with the yeast 80 kDa subunit, which was earlier suggested to be involved in S phase cell cycle control in a pathway that is able to sense and signal incomplete replication. The small subunits of human and mouse DNA polymerase epsilon also show homology to the C-terminal domain of the second largest subunit of DNA polymerase alpha. The gene for the small subunit of human DNA polymerase epsilon (POLE2) was localized to chromosome 14q21-q22 by fluorescence in situ hybridization.
Assuntos
DNA Polimerase II/química , DNA Polimerase II/genética , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Cromossomos Humanos Par 14/genética , Clonagem Molecular , DNA Complementar/genética , Humanos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
This paper describes a new system in which optical reflectance is used to test the pulse and vitality of oral mucosa or dental pulp. Radiation at red (660 nm) and near infra-red (850 nm) wavelengths are directed through a thin probe. The beam is directed into tissue and reflected back. Plethysmography is used to measure the pulse rate from the right forefinger. Reflected radiation is related to plethysmogram using a computer. Preliminary findings relating to the lips and gingiva in 9 healthy volunteers were promising. Preliminary tests showed that vital and nonvital pulps reflected the radiation differently. Pulpal pulse did not always correspond to plethysmogram from the right forefinger.