Assuntos
Bactérias/enzimologia , Metilases de Modificação do DNA/metabolismo , DNA/metabolismo , Oligodesoxirribonucleotídeos/isolamento & purificação , S-Adenosilmetionina/metabolismo , Sequência de Bases , Cromatografia/métodos , Cromatografia em Camada Fina/métodos , Clonagem Molecular , DNA/química , DNA/genética , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Radioisótopos de Fósforo , Técnica de Diluição de Radioisótopos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Radioisótopos de Enxofre , TrítioRESUMO
The genes for FokI, a type-IIS restriction-modification system from Flavobacterium okeanokoites (asymmetric recognition sequence: 5'-GGATG/3'-CCTAC), were cloned into Escherichia coli. Recombinants carrying the fokIR and fokIM genes were found to modify their DNA completely, and to restrict lambdoid phages weakly. The nt sequences of the genes were determined, and the probable start codons were confirmed by aa sequencing. The FokI endonuclease (R.FokI) and methyltransferase (M.FokI) are encoded by single, adjacent genes, aligned in the same orientation, in the order M then R. The genes are large by the standards of type-II systems, 1.9 kb for the M gene, and 1.7 kb for the R gene. Preceding each gene is a pair of FokI recognition sites; it is conceivable that interactions between the sites and the FokI proteins could regulate expression of the genes. The aa sequences of the N- and C-terminal halves of M.FokI are similar to one another, and to certain other DNA-adenine methyltransferases, suggesting that the enzyme has a 'tandem' structure, such as could have arisen by the fusion of a pair of adjacent, ancestral M genes. Truncated derivatives of M. FokI were constructed by deleting the 5'- or 3'-ends of the fokIM gene. Deleting most of the C-terminus of M.FokI produced derivatives that methylated only the top (GGATG) strand of the recognition sequence. Conversely, deleting most of the N-terminus produced derivatives that methylated only the bottom (CATCC) strand of the recognition sequence. These results indicate that the domains in M.FokI for methylating the two strands of the recognition sequence are largely separate.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Flavobacterium/enzimologia , Metiltransferases/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Análise Mutacional de DNA , Escherichia coli/genética , Flavobacterium/genética , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
M.FokI, a type-IIS modification enzyme from Flavobacterium okeanokoites, was purified, and its activity was characterized in vitro. The enzyme was found to be a DNA-adenine methyltransferase and to methylate both strands of the asymmetric FokI recognition sequence: (formula; see text) M.FokI does not methylate single-stranded DNA, nor does it methylate double-stranded DNA at sequences other than FokI sites.