RESUMO
A new type II restriction endonuclease designated PfoI has been partially purified from Pseudomonas fluorescens biovar 126. PfoI recognises the interrupted hexanucleotide palindromic sequence 5'-T downward arrow CCNGGA-3' and cleaves DNA to produce protruding pentanucleotide 5'-ends.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Pseudomonas fluorescens/enzimologia , Sequência de Bases , Sítios de Ligação/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/efeitos dos fármacos , Desoxirribonucleases de Sítio Específico do Tipo II/isolamento & purificação , Concentração de Íons de Hidrogênio , Magnésio/farmacologia , Análise de Sequência de DNA , TemperaturaRESUMO
We report the properties of the new AloI restriction and modification enzyme from Acinetobacter lwoffi Ks 4-8 that recognizes the DNA target 5' GGA(N)6GTTC3' (complementary strand 5' GAAC(N)6TCC3'), and the nucleotide sequence of the gene encoding this enzyme. AloI is a bifunctional large polypeptide (deduced M(r) 143 kDa) revealing both DNA endonuclease and methyltransferase activities. Depending on reaction cofactors, AloI cleaves double-stranded DNA on both strands, seven bases on the 5' side, and 12-13 bases on the 3' side of its recognition sequence, and modifies adenine residues in both DNA strands in the target sequence yielding N6-methyladenine. For cleavage activity AloI maintains an absolute requirement for Mg(2+) and does not depend on or is stimulated by either ATP or S-adenosyl-L-methionine. Modification function requires the presence of S-adenosyl-L-methionine and is stimulated by metal ions (Ca(2+)). The C-terminal and central parts of the protein were found to be homologous to certain specificity (HsdS) and modification (HsdM) subunits of type I R-M systems, respectively. The N-terminal part of the protein possesses the putative endonucleolytic motif DXnEXK of restriction endonucleases. The deduced amino acid sequence of AloI shares significant homology with polypeptides encoding HaeIV and CjeI restriction-modification proteins at the N-terminal and central, but not at the C-terminal domains. The organization of AloI implies that its evolution involved fusion of an endonuclease and the two subunits, HsdM and HsdS, of type I restriction enzymes. According to the structure and function properties AloI may be regarded as one more representative of a newly emerging group of HaeIV-like restriction endonucleases. Discovery of these enzymes opens new opportunities for constructing restriction endonucleases with a new specificity.
Assuntos
Acinetobacter/enzimologia , Metilação de DNA , Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/metabolismo , Acinetobacter/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Cromatografia em Gel , Clonagem Molecular , Coenzimas/metabolismo , Metilases de Modificação do DNA/isolamento & purificação , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/isolamento & purificação , Concentração de Íons de Hidrogênio , Magnésio/metabolismo , Dados de Sequência Molecular , Peso Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Concentração Osmolar , Estrutura Terciária de Proteína , S-Adenosilmetionina/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
We report the properties of the new BseMII restriction and modification enzymes from Bacillus stearothermophilus Isl 15-111, which recognize the 5'-CTCAG sequence, and the nucleotide sequence of the genes encoding them. The restriction endonuclease R.BseMII makes a staggered cut at the tenth base pair downstream of the recognition sequence on the upper strand, producing a two base 3'-protruding end. Magnesium ions and S:-adenosyl-L-methionine (AdoMet) are required for cleavage. S:-adenosylhomocysteine and sinefungin can replace AdoMet in the cleavage reaction. The BseMII methyltransferase modifies unique adenine residues in both strands of the target sequence 5'-CTCAG-3'/5'-CTGAG-3'. Monomeric R.BseMII in addition to endonucleolytic activity also possesses methyltransferase activity that modifies the A base only within the 5'-CTCAG strand of the target duplex. The deduced amino acid sequence of the restriction endonuclease contains conserved motifs of DNA N6-adenine methylases involved in S-adenosyl-L-methionine binding and catalysis. According to its structure and enzymatic properties, R.BseMII may be regarded as a representative of the type IV restriction endonucleases.
