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Kinetics of Methylation by EcoP1I DNA Methyltransferase.

Bheemanaik S, Sistla S, Krishnamurthy V, Arathi S, Desirazu NR - Enzyme Res (2010)

Bottom Line: M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits.EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site.A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.

ABSTRACT
EcoP1I DNA MTase (M.EcoP1I), an N(6)-adenine MTase from bacteriophage P1, is a part of the EcoP1I restriction-modification (R-M) system which belongs to the Type III R-M system. It recognizes the sequence 5'-AGACC-3' and methylates the internal adenine. M.EcoP1I requires Mg(2+) for the transfer of methyl groups to DNA. M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits. Preincubation and isotope partitioning studies with M.EcoP1I indicate a kinetic mechanism where the duplex DNA binds first followed by AdoMet. Interestingly, M.EcoP1I methylates DNA substrates in the presence of Mn(2+) and Ca(2+) other than Mg(2+) with varying affinities. Amino acid analysis and methylation assays in the presence of metal ions suggest that M.EcoP1I has indeed two metal ion-binding sites [(358)ID(x)(n) … ExK(401) and (600)DxDxD(604) motif]. EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site. A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.

No MeSH data available.


Distributive mode of methylation catalyzed by M.EcoP1I and M.EcoP15I. (a) M.EcoP1I (250 nM) was preincubated at room temperature with 2 μM of biotin-tagged oligonucleotide (duplex II) containing two EcoP1I recognition sites for 5 minutes. After preincubation, the mixture was divided into two sets and to one set; [3H-methyl]AdoMet was added to start the reaction (∙). To the other set, [3H-methyl]AdoMet was added along with 20 μM of nonbiotin-tagged oligonucleotide (duplex III) to chase the methylation reaction (∘). The reaction was monitored at different time intervals and it indicated and analyzed the extent of methylation of biotin-tagged duplex II using biotin-avidin micro plate assay. (b) M.EcoP15I (250 nM) was preincubated at room temperature with 2 μM of biotin-tagged oligonucleotide (duplex II) containing two EcoP15I recognition sites for 5 minutes. The methylation reaction was performed as described in Materials and Methods and monitored the reaction at different time intervals for methylation of biotin-tagged oligonucleotide (duplex II).
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fig6: Distributive mode of methylation catalyzed by M.EcoP1I and M.EcoP15I. (a) M.EcoP1I (250 nM) was preincubated at room temperature with 2 μM of biotin-tagged oligonucleotide (duplex II) containing two EcoP1I recognition sites for 5 minutes. After preincubation, the mixture was divided into two sets and to one set; [3H-methyl]AdoMet was added to start the reaction (∙). To the other set, [3H-methyl]AdoMet was added along with 20 μM of nonbiotin-tagged oligonucleotide (duplex III) to chase the methylation reaction (∘). The reaction was monitored at different time intervals and it indicated and analyzed the extent of methylation of biotin-tagged duplex II using biotin-avidin micro plate assay. (b) M.EcoP15I (250 nM) was preincubated at room temperature with 2 μM of biotin-tagged oligonucleotide (duplex II) containing two EcoP15I recognition sites for 5 minutes. The methylation reaction was performed as described in Materials and Methods and monitored the reaction at different time intervals for methylation of biotin-tagged oligonucleotide (duplex II).

Mentions: To determine the mode of methylation by M.EcoP1I, a 51 mer oligonucleotide substrate (duplex II) with a biotin-tag at the 5′ end that contained two EcoP1I recognition sites separated by 19 bp was used. The biotin-tagged oligonucleotide (2 μM) was incubated with M.EcoP1I (250 nM) for 5 minutes at room temperature. After the preincubation, the reaction mixture was split in two, to one half, 1 μM of [3H-methyl]AdoMet was added to follow the methyl transfer reaction up to 15 minutes. To the other half, 1 μM of [3H-methyl]AdoMet and 20 μM of a 51 mer oligonucleotide substrate (duplex III) without a biotin-tag as a trap was followed by methylation up to 15 minutes. The methylation of the biotin-tagged oligonucleotide (duplex II) was followed by a biotin-avidin micro plate assay (Figure 6(a)). In the presence of a 10-fold excess of a nonbiotin-tagged oligonucleotide, the extent of methylation of the biotin-tagged oligonucleotides did not increase, but in the absence of nonbiotin-tagged oligonucleotides, an increase in methylation was observed (Figure 6(a)). To rule out any inhibition of an M.EcoP1I catalyzed methylation reaction in the presence of an excess of DNA, a filter binding assay was carried out with a reaction mixture containing both biotin-tagged and nonbiotin-tagged oligonucleotides. The rate of methylation was almost similar in both conditions suggesting that there was no substrate inhibition in the presence of an excess of DNA (data not shown). These results can be explained only if the DNA is modified by the distributive mode of action. During the distributive mode of the methylation reaction, the enzyme binds to DNA and dissociates from the DNA molecule after one round of catalysis.


Kinetics of Methylation by EcoP1I DNA Methyltransferase.

Bheemanaik S, Sistla S, Krishnamurthy V, Arathi S, Desirazu NR - Enzyme Res (2010)

Distributive mode of methylation catalyzed by M.EcoP1I and M.EcoP15I. (a) M.EcoP1I (250 nM) was preincubated at room temperature with 2 μM of biotin-tagged oligonucleotide (duplex II) containing two EcoP1I recognition sites for 5 minutes. After preincubation, the mixture was divided into two sets and to one set; [3H-methyl]AdoMet was added to start the reaction (∙). To the other set, [3H-methyl]AdoMet was added along with 20 μM of nonbiotin-tagged oligonucleotide (duplex III) to chase the methylation reaction (∘). The reaction was monitored at different time intervals and it indicated and analyzed the extent of methylation of biotin-tagged duplex II using biotin-avidin micro plate assay. (b) M.EcoP15I (250 nM) was preincubated at room temperature with 2 μM of biotin-tagged oligonucleotide (duplex II) containing two EcoP15I recognition sites for 5 minutes. The methylation reaction was performed as described in Materials and Methods and monitored the reaction at different time intervals for methylation of biotin-tagged oligonucleotide (duplex II).
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Related In: Results  -  Collection

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fig6: Distributive mode of methylation catalyzed by M.EcoP1I and M.EcoP15I. (a) M.EcoP1I (250 nM) was preincubated at room temperature with 2 μM of biotin-tagged oligonucleotide (duplex II) containing two EcoP1I recognition sites for 5 minutes. After preincubation, the mixture was divided into two sets and to one set; [3H-methyl]AdoMet was added to start the reaction (∙). To the other set, [3H-methyl]AdoMet was added along with 20 μM of nonbiotin-tagged oligonucleotide (duplex III) to chase the methylation reaction (∘). The reaction was monitored at different time intervals and it indicated and analyzed the extent of methylation of biotin-tagged duplex II using biotin-avidin micro plate assay. (b) M.EcoP15I (250 nM) was preincubated at room temperature with 2 μM of biotin-tagged oligonucleotide (duplex II) containing two EcoP15I recognition sites for 5 minutes. The methylation reaction was performed as described in Materials and Methods and monitored the reaction at different time intervals for methylation of biotin-tagged oligonucleotide (duplex II).
Mentions: To determine the mode of methylation by M.EcoP1I, a 51 mer oligonucleotide substrate (duplex II) with a biotin-tag at the 5′ end that contained two EcoP1I recognition sites separated by 19 bp was used. The biotin-tagged oligonucleotide (2 μM) was incubated with M.EcoP1I (250 nM) for 5 minutes at room temperature. After the preincubation, the reaction mixture was split in two, to one half, 1 μM of [3H-methyl]AdoMet was added to follow the methyl transfer reaction up to 15 minutes. To the other half, 1 μM of [3H-methyl]AdoMet and 20 μM of a 51 mer oligonucleotide substrate (duplex III) without a biotin-tag as a trap was followed by methylation up to 15 minutes. The methylation of the biotin-tagged oligonucleotide (duplex II) was followed by a biotin-avidin micro plate assay (Figure 6(a)). In the presence of a 10-fold excess of a nonbiotin-tagged oligonucleotide, the extent of methylation of the biotin-tagged oligonucleotides did not increase, but in the absence of nonbiotin-tagged oligonucleotides, an increase in methylation was observed (Figure 6(a)). To rule out any inhibition of an M.EcoP1I catalyzed methylation reaction in the presence of an excess of DNA, a filter binding assay was carried out with a reaction mixture containing both biotin-tagged and nonbiotin-tagged oligonucleotides. The rate of methylation was almost similar in both conditions suggesting that there was no substrate inhibition in the presence of an excess of DNA (data not shown). These results can be explained only if the DNA is modified by the distributive mode of action. During the distributive mode of the methylation reaction, the enzyme binds to DNA and dissociates from the DNA molecule after one round of catalysis.

Bottom Line: M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits.EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site.A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.

ABSTRACT
EcoP1I DNA MTase (M.EcoP1I), an N(6)-adenine MTase from bacteriophage P1, is a part of the EcoP1I restriction-modification (R-M) system which belongs to the Type III R-M system. It recognizes the sequence 5'-AGACC-3' and methylates the internal adenine. M.EcoP1I requires Mg(2+) for the transfer of methyl groups to DNA. M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits. Preincubation and isotope partitioning studies with M.EcoP1I indicate a kinetic mechanism where the duplex DNA binds first followed by AdoMet. Interestingly, M.EcoP1I methylates DNA substrates in the presence of Mn(2+) and Ca(2+) other than Mg(2+) with varying affinities. Amino acid analysis and methylation assays in the presence of metal ions suggest that M.EcoP1I has indeed two metal ion-binding sites [(358)ID(x)(n) … ExK(401) and (600)DxDxD(604) motif]. EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site. A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.

No MeSH data available.