RESUMO
DNA ligase of E. coli is a polypeptide of molecular weight 75,000. The comparable T4-induced enzyme is somewhat smaller (63,000 to 68,000). Both enzymes catalyze the synthesis of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl groups in nicked duplex DNA, coupled to the cleavage of the pyrophosphate bond of DPN (E. coli) or ATP (T4). Phosphodiester bond synthesis catalyzed by both enzymes occurs in a series of these discrete steps and involves the participation of two covalent intermediates (Fig. 1). A steady state kinetic analysis of the reaction-catalyzed E. coli ligase supports this mechanism, and further demonstrates that enzyme-adenylate and DNA-adenylate are kinetically significant intermediates on the direct path of phosphodiester bond synthesis. A strain of E. coli with a mutation in the structural gene for DNA ligase which results in the synthesis of an abnormally thermolabile enzyme is inviable at 42 degrees C. Although able to grow at 30 degrees C, the mutant is still defective at this temperature in its ability to repair damage to its DNA caused by ultraviolet irradiation and by alkylating agents. At 42 degrees C, all the newly replicated DNA is in the form of short 10S "Okazaki fragments," an indication that the reason for the mutant's failure to survive under these conditions is its inability to sustain the ligation step that is essential for the discontinuous synthesis of the E. coli chromosome. DNA ligase is therefore an essential enzyme required for normal DNA replication and repair in E. coli. Purified DNA ligases have proved to be useful reagents in the construction in vitro of recombinant DNA molecules.
Assuntos
Escherichia coli/enzimologia , Polinucleotídeo Ligases , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Colífagos/enzimologia , DNA/metabolismo , Reparo do DNA , Replicação do DNA , DNA Circular/metabolismo , Cinética , Modelos Químicos , Peso Molecular , Mutação , NAD/metabolismo , Polinucleotídeo Ligases/metabolismo , Conformação Proteica , Recombinação GenéticaRESUMO
A lambda vector that contains the gene for Escherichia coli DNA ligase (lambdagt4-lop-11 lig+) has been modified to achieve overproduction of this enzyme. The third Eco RI site in the lambda chromosome has been altered by mutation, and the left-hand Eco RI fragment has been shortened. The new vector, lambdagt4-lop-11 lig+, forms a stable lysogen which, upon induction, produces a 100-fold increase in DNA ligase activity. Introduction of a phage mutation (S7) that prevents cell lysis results in an even greater increase (500-fold).
Assuntos
Colífagos/metabolismo , DNA Recombinante/metabolismo , DNA Viral/metabolismo , Polinucleotídeo Ligases/biossíntese , DNA Bacteriano/metabolismo , Escherichia coli , Genes , Lisogenia , MutaçãoRESUMO
The herpes simplex virus type 1 encoded ICP8 protein binds single-stranded (ss) DNA and is required for DNA replication in vitro. We have used electron microscopy to examine the ability of ICP8 to promote homologous pairing and strand transfer reactions. Visualization of M13 ssDNA-ICP8 complexes showed that they preferentially bound and enveloped homologous double-stranded (ds) DNA fragments; their deproteinization released ssDNA circles containing dsDNA segments, and an equal number of linear single strands. Optimal transfer required Mg2+ but not nucleoside triphosphates, and showed a fourfold preference for dsDNA fragments with a few bases recessed ends. Gel electrophoretic analysis confirmed the strand transfer activity of ICP8.
Assuntos
DNA de Cadeia Simples/genética , DNA Viral/genética , Recombinação Genética , Simplexvirus/genética , Proteínas Virais/metabolismo , DNA Circular/genética , DNA de Cadeia Simples/ultraestrutura , DNA Viral/ultraestrutura , Proteínas de Ligação a DNA , Magnésio/farmacologia , Recombinação Genética/efeitos dos fármacos , Homologia de Sequência do Ácido Nucleico , Endonucleases Específicas para DNA e RNA de Cadeia Simples/farmacologiaRESUMO
UL9 protein and ICP8 encoded by the herpes simplex virus type 1 (HSV-1) were shown to catalyze a highly active, non-origin-dependent unwinding of DNA. UL9 protein, the HSV-1 origin binding protein, as a modest helicase activity that is greatly stimulated by the HSV-1 single strand (ss) binding protein, ICP8. Here, electron microscopy has been applied to examine the mechanics of this reaction. Negative staining of the proteins revealed particles consisting primarily of ICP8 monomers and UL9 protein dimers. When the binding of UL9 protein to double strand (ds) DNA containing ss tails was examined by shadowcasting methods, UL9 protein was seen bound to the ss tails or ss/ds junctions; addition of ATP led to its appearance internally along the ds segment. When UL9 protein and ICP8 were incubated together with the tailed dsDNA in the presence of ATP, a highly ordered unwinding of the DNA was observed by negative staining that appeared to progress through four distinct stages: (1) binding of ICP8 to the ss tail and progressive coverage of the ds portion by UL9 protein; (2) formation of highly condensed regular filaments; (3) relaxation of the condensed structures into coiled-coils; and (4) unwinding of the coils and release of ICP8-covered linear ssDNAs. This process represents a mechanism of unwinding that is very different from ones that proceed by a progressive unwinding at Y-shaped forks that move along the DNA.
Assuntos
DNA Helicases/fisiologia , DNA Viral/ultraestrutura , Proteínas de Ligação a DNA/fisiologia , Microscopia Eletrônica , Conformação de Ácido Nucleico , Simplexvirus/genética , Proteínas Virais/fisiologia , Trifosfato de Adenosina/farmacologia , DNA/ultraestrutura , DNA Helicases/ultraestrutura , DNA de Cadeia Simples/ultraestrutura , DNA Viral/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Coloração Negativa , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/ultraestrutura , Técnica Histológica de Sombreamento , Proteínas Virais/ultraestruturaRESUMO
The cDNA encoding the catalytic subunit of Drosophila melanogaster (Dm) DNA polymerase delta (Pol delta) was isolated by a combination of PCR amplification and cDNA library screening. The cDNA is 3457 nucleotides in length and contains an open reading frame (ORF) that encodes a protein of 1092 amino acids (124,799 Da). The ORF contains the sequence that was determined for a peptide from the purified catalytic subunit of Dm Pol delta. Polyclonal antibodies raised against Dm Pol delta specifically recognize a protein of the expected size when the cDNA is expressed in either Escherichia coli or insect cells. Comparison of the deduced aa sequence with other Pol delta sequences demonstrates that Pol delta is one of the most highly conserved of the DNA polymerases.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Polimerase III , DNA Complementar/genética , Expressão Gênica , Genes de Insetos , Dados de Sequência Molecular , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Herpes simplex virus type 1 (HSV1) encodes a heterotrimeric helicase-primase comprised of the products of three of the seven DNA replication-specific genes. Several dihalo-substituted derivatives of N2-phenylguanines and 2-anilinoadenines weakly inhibited the intrinsic DNA-dependent NTPase activity of the HSV1 helicase-primase, and these compounds inhibited the DNA-unwinding activity of the enzyme. The primase activity of the enzyme was strongly inhibited by 3,4- and 3,5-dichloroanilino derivatives of adenine and 2-aminopyrimidines. These compounds and nucleoside analogs of 2-(3,5-dichloroanilino)purines inhibited viral DNA synthesis in HSV1-infected HeLa cells in culture but also inhibited cellular DNA synthesis, likely as a result of inhibition of cellular primase and/or DNA polymerases.