DNA recognition by the EcoP15I and EcoPI modification methyltransferases

Ahmad, Ishtiyaque; Krishnamurthy, Vinita; Rao, Desirazu N.

doi:10.1016/0378-1119(95)00671-r

Cited by 32 publications

(23 citation statements)

References 11 publications

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“…We employed size-exclusion chromatography and dimethylsuberimidate crosslinking of the subunits. Previously, we showed that the wild-type M.EcoP15I exists as a dimer (Ahmad et al, 1995). The elution profiles of the mutant G448S and the wild-type M.EcoP15I were superimposable (data not shown).…”

Section: Mutant Mtases Exist As a Dimermentioning

confidence: 76%

Functional Analysis of Conserved Motifs inEcoP15I DNA Methyltransferase

Ahmad

Rao

1996

Journal of Molecular Biology

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Section: Mutant Mtases Exist As a Dimermentioning

confidence: 76%

Functional Analysis of Conserved Motifs inEcoP15I DNA Methyltransferase

Ahmad

Rao

1996

Journal of Molecular Biology

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“…It was reported that DMS-treated proteins could migrate as a diffuse smear. This smearing arises because of nonuniform cross-linking, which can lead to differential binding of SDS (42,43). Because of the large number of lysine residues, it is not surprising that more than one cross-linked species would be present.…”

Section: Determination Of the Oligomeric State Of Ccrmmentioning

confidence: 99%

Identification of the Active Oligomeric State of an Essential Adenine DNA Methyltransferase from Caulobacter crescentus

Shier

Hancey

Benkovic

2001

Journal of Biological Chemistry

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Caulobacter crescentus contains one of the two known prokaryotic DNA methyltransferases that lacks a cognate endonuclease. This endogenous cell cycle regulated adenine DNA methyltransferase (CcrM) is essential for C. crescentus cellular viability. DNA methylation catalyzed by CcrM provides an obligatory signal for the proper progression through the cell cycle. To further our understanding of the regulatory role played by CcrM, we sought to investigate its biophysical properties. In this paper we employed equilibrium ultracentrifugation, velocity ultracentrifugation, and chemical cross-linking to show that CcrM is dimeric at physiological concentrations. However, surface plasmon resonance experiments in the presence of S-adenosyl-homocysteine evince that CcrM binds as a monomer to a defined hemi-methylated DNA substrate containing the canonical methylation sequence, GANTC. Initial velocity experiments demonstrate that dimerization of CcrM does not affect DNA methylation. Collectively, these findings suggest that CcrM is active as a monomer and provides a possible in vivo role for dimerization as a means to stabilize CcrM from premature catabolism.

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“…8A). We had earlier reported that the wild-type enzyme exists as a dimer of molecular mass 150,000 Da in solution (6,7). We determined the oligomeric nature of the mutant enzymes by employing glutaraldehyde cross-linking of the subunits.…”

Section: Interaction Of Mutant Ecop15i Dna Methyltransferases With Olmentioning

confidence: 99%

“…We had earlier shown by gel mobility shift assays that EcoP15I DNA MTase binds about 3-fold more tightly to DNA containing its recognition sequence 5Ј-CAG-CAG-3Ј than to nonspecific sequences in the absence or presence of cofactors. Interestingly, in the presence of ATP, the discrimination between specific and nonspecific sequences increased significantly (6,7).…”

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Probing the Role of Cysteine Residues in the EcoP15I DNA Methyltransferase

Reddy

Rao

1998

Journal of Biological Chemistry

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Chemical modification using thiol-directed agents and site-directed mutagenesis has been used to investigate the role of cysteine residues of EcoP15I DNA methyltransferase. Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethylmaleimide and iodoacetamide. 5,5-Dithiobis(2-nitrobenzoic acid) titration of the enzyme under nondenaturing and denaturing conditions confirmed the presence of six cysteine residues without any disulfides in the protein. Aware that relatively bulky reagents inactivate the methyltransferase by directly occluding the substrate-binding site or by locking the methyltransferase in an inactive conformation, we used site-directed mutagenesis to sequentially replace each of the six cysteines in the protein at positions 30, 213, 344, 434, 553, and 577. All the resultant mutant methylases except for the C344S and C344A enzymes retained significant activity as assessed by in vivo and in vitro assays. The effects of the substitutions on the function of EcoP15I DNA methyltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis. Our results clearly indicate that the cysteines at positions other than 344 are not essential for activity. In contrast, the C344A enzyme showed a marked loss of enzymatic activity. More importantly, whereas the inactive C344A mutant enzyme bound S-adenosyl-L-methionine, it failed to bind to DNA. Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive. Taken together, these results convincingly demonstrate that the Cys-344 is necessary for enzyme activity and indicate an essential role for it in DNA binding.EcoP15I DNA methyltransferase (EcoP15I DNA MTase) 1 catalyzes the transfer of a methyl group from S-adenosyl-Lmethionine (AdoMet) to the second adenine nucleotide in the canonical site 5Ј-CAGCAG-3Ј (1) to form N 6 -methyladenine. The enzyme is part of the type III restriction-modification (R-M) system (2). Type III R-M enzymes are multifunctional proteins that exert both methylation and restriction activities (2). Type III R-M systems contain two subunits, the Res subunit encoded by the res gene and the Mod subunit encoded by the mod gene. Although the Mod subunit alone can catalyze the methylation reaction, both the Res and Mod subunits are necessary for DNA cleavage (2). The enzymes have an absolute requirement for ATP for restriction, and recently we and others (3, 4) showed that ATP hydrolysis was required for DNA cleavage. It has been shown that only the Mod subunit is involved in DNA sequence recognition in both the restriction and modification reactions (5). We had earlier shown by gel mobility shift assays that EcoP15I DNA MTase binds about 3-fold more tightly to DNA containing its recognition sequence 5Ј-CAG-CAG-3Ј than to nonspecific sequences in the absence or presence of cofactors. Interestingly, in the presence ...

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DNA recognition by the EcoP15I and EcoPI modification methyltransferases

Cited by 32 publications

References 11 publications

Functional Analysis of Conserved Motifs inEcoP15I DNA Methyltransferase

Functional Analysis of Conserved Motifs inEcoP15I DNA Methyltransferase

Identification of the Active Oligomeric State of an Essential Adenine DNA Methyltransferase from Caulobacter crescentus

Probing the Role of Cysteine Residues in the EcoP15I DNA Methyltransferase

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