Assuntos
Proteína ADAM17/metabolismo , Vesículas Extracelulares/virologia , Infecções por HIV/virologia , Interações Hospedeiro-Patógeno/fisiologia , Proteínas Proto-Oncogênicas c-hck/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Proteína do Núcleo p24 do HIV/metabolismo , Infecções por HIV/patologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Masculino , Células Mieloides/metabolismo , Células Mieloides/virologia , Proteínas Proto-Oncogênicas c-hck/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
DNA polymerase epsilon (Pol epsilon) is believed to play an essential catalytic role during eukaryotic DNA replication and is thought to participate in recombination and DNA repair. That Pol epsilon is essential for progression through S phase and for viability in budding and fission yeasts is a central element of support for that view. We show that the amino-terminal portion of budding yeast Pol epsilon (Pol2) containing all known DNA polymerase and exonuclease motifs is dispensable for DNA replication, DNA repair, and viability. However, the carboxy-terminal portion of Pol2 is both necessary and sufficient for viability. Finally, the viability of cells lacking Pol2 catalytic function does not require intact DNA replication or damage checkpoints.
Assuntos
DNA Polimerase II/metabolismo , Reparo do DNA , Replicação do DNA , Leveduras/genética , Alelos , Domínio Catalítico , DNA Polimerase II/química , DNA Fúngico/análise , Regulação Fúngica da Expressão Gênica , Mutagênese/fisiologia , Regiões Promotoras Genéticas/fisiologia , Estrutura Terciária de Proteína , Leveduras/citologia , Leveduras/enzimologiaRESUMO
The catalytic subunit of human DNA polymerase epsilon, an enzyme involved in nuclear DNA replication and repair, is encoded by the POLE1 gene. This gene is composed of 51 exons spanning at least 97 kb of genomic DNA. It was found to encode three alternative mRNA splice variants that differ in their 5'-terminal sequences and in the N-termini of the predicted proteins. A CpG island covers the promoter region for the major transcript in HeLa cells. This promoter is TATA-less and contains several putative binding sites for transcription factors typical of S-phase-up-regulated and serum-responsive promoters. Potential promoter regions were also identified for the two other alternative transcripts. Interestingly, no nuclear polyadenylation signal sequence was detected in the 3'-untranslated region, although a poly(A) tail was present. These results suggest a complicated regulatory machinery for the expression of the human POLE1 gene, including three alternative transcripts expressed from three promoters.
Assuntos
Processamento Alternativo , Domínio Catalítico/genética , DNA Polimerase II/genética , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Cromossomos Humanos Par 12/genética , Clonagem Molecular , Códon de Iniciação/genética , Ilhas de CpG/genética , Éxons/genética , Dosagem de Genes , Biblioteca Gênica , Células HeLa , Humanos , Íntrons/genética , Dados de Sequência Molecular , Poli A/genética , Proteínas de Ligação a Poli-ADP-Ribose , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , TATA Box/genéticaRESUMO
DNA polymerase epsilon [DNA pol epsilon (EC 2.7.7.7)] is one of the four nuclear DNA polymerases in eukaryotic cells. The mammalian enzyme is involved in DNA repair and possibly also in replication of chromosomal DNA. The gene encoding pol epsilon (POLE) was assigned to human and rat chromosomes 12 by Southern blot analysis of genomic DNA from mouse-human and mouse-rat somatic cell hybrid panels using human cDNA probe. The human gene was then regionally localized to band 12q24.3 by fluorescence in situ hybridization of metaphase spreads of chromosomes from human lymphocytes using genomic DNA probe. POLE is closely linked to HNF1A, a gene encoding a liver-enriched transcription factor, HNF1 alpha. The two genes thus define a new synteny group retained on human and rat chromosomes 12. Another gene mapping to the same or close-by region as human POLE is a gene for inherited disorder tuberous sclerosis 3, TSC3.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 12 , DNA Polimerase Dirigida por DNA/genética , Animais , DNA Polimerase II , Reparo do DNA , Replicação do DNA , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , RatosRESUMO
The cDNA encoding the catalytic polypeptide of human DNA polymerase epsilon was cloned. The deduced amino acid sequence reveals that the catalytic polypeptide is 2257 amino acids in length and its calculated molecular mass is 258 kDa. A single RNA message of 7.5 kilobases was recognized by isolated cDNA clones. The identity of the cDNA was verified by direct amino acid sequencing of tryptic fragments derived from the catalytic polypeptide of the HeLa DNA polymerase epsilon. The primary structure comparison with multiple DNA polymerases indicates that human DNA polymerase epsilon catalytic polypeptide is a homolog of the yeast Saccharomyces cerevisiae DNA polymerase II catalytic polypeptide. The proteins are 39% identical. In the region containing known DNA polymerase consensus motifs, the identity is 63%. The expression of the mRNA encoding DNA polymerase epsilon is strongly dependent on cell proliferation.
Assuntos
DNA de Neoplasias/genética , DNA Polimerase Dirigida por DNA/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Northern Blotting , Clonagem Molecular/métodos , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Sondas de DNA , DNA de Neoplasias/isolamento & purificação , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Células HeLa , Humanos , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , Vírus/genéticaRESUMO
The 3'-->5' exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3' end of an oligonucleotide with a non-processive mechanism and leaves 5'-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3' end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slowed down when double-stranded region is reached. The preferential removal of a non-complementary 3' end and the nonprocessive mechanism are consistent with anticipated proofreading function. In addition to the 3'-->5' exonuclease activity, an 5'-->3' exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential component for the action of this enzyme in vivo. Contrary to the 3'-->5' exonuclease activity, the 5'-->3' exonuclease is separable from the polymerase activity.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Exonucleases/metabolismo , Sequência de Bases , DNA Polimerase II , DNA Polimerase III , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/isolamento & purificação , Células HeLa , Humanos , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen-independent form of DNA polymerase delta, has been shown elsewhere to be catalytically and structurally distinct from DNA polymerase delta. The catalytic activity of HeLa DNA polymerase epsilon, an enzyme consisting of greater than 200- and 55-kDa polypeptides, was assigned to the larger polypeptide by polymerase trap reaction. This catalytic polypeptide was cleaved by incubation with trypsin into two polypeptide fragments with molecular masses of 122 and 136 kDa, the former of which was relatively resistant to further proteolysis and possessed the polymerase activity. The cleavage increased the polymerase and exonuclease activities of the enzyme some 2-3-fold. DNA polymerase epsilon was also purified in a smaller 140-kDa form from calf thymus. The digestion of this form of the enzyme by trypsin also generated a 122-kDa polypeptide. These results suggest that the catalytic core of DNA polymerase epsilon is a 258-kDa polypeptide that is composed of two segments linked with a protease-sensitive area. One of the segments harbors both DNA polymerase and 3'----5' exonuclease activities. In spite of the different polypeptide structures, the catalytic properties of the HeLa enzyme, its trypsin-digested form, and the calf thymus enzyme remained essentially the same.