Interaction of EcoP15I DNA Methyltransferase with Oligonucleotides Containing the Asymmetric Sequence 5′-CAGCAG-3′

Ahmad, Ishtiyaque; Rao, Desirazu N.

doi:10.1006/jmbi.1994.1588

Cited by 39 publications

(43 citation statements)

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“…Methylation Assay-Methylation assays monitored incorporation of tritiated methyl groups into DNA by using a modified ion-exchange filter assay (16). Methylation assays were carried out in a reaction mixture containing 300 nM supercoiled pUC18 DNA, 1 M [methyl-…”

Section: Methodsmentioning

confidence: 99%

“…Electrophoresis was performed as described (16). Gels were dried and exposed to x-ray film at room temperature for 16 -20 h. Gel Filtration Chromatography-Native molecular masses of wild type and M.EcoP15I-M357P were determined by gel filtration chromatography using a calibrated Superdex 200 column.…”

Section: Methodsmentioning

confidence: 99%

“…Increasing the (Table 1) in binding buffer for 10 min on ice. Electrophoresis was performed as described (16). The gel was dried and exposed to x-ray film at room temperature for 16 -20 h. C, photolabeling of wild type and mutant EcoP15I DNA MTases with [methyl-3 H]AdoMet.…”

Section: Site-directed Mutagenesis Expression and Purification Of Mmentioning

confidence: 99%

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A Mutation in the Mod Subunit of EcoP15I Restriction Enzyme Converts the DNA Methyltransferase to a Site-specific Endonuclease

Bist¹,

Madhusoodanan²,

Rao

2007

Journal of Biological Chemistry

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A closer inspection of the amino acid sequence of EcoP15I DNA methyltransferase revealed a region of similarity to the PDX n (D/E)XK catalytic site of type II restriction endonucleases, except for methionine in EcoP15I DNA methyltransferase instead of proline. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. EcoP15I-M357P DNA methyltransferase specifically binds to the recognition sequence 5-CAGCAG-3 and cleaves DNA asymmetrically EcoP151-M357P⅐DNA methyltransferase specifically binds to the recognition sequence 5-CAGCAG-3 and cleaves DNA asymmetrically, 5-CAG-CAG(N) 10 -3, as indicated by the arrows, in presence of magnesium ions.Sequence discrimination by DNA-binding proteins is one of the most remarkable examples of molecular recognition in biochemistry. Restriction-modification (R-M) 4 systems are paradigms for the study of protein-DNA recognition. More than 3600 R-M enzymes have been identified so far, and they manifest 218 unique recognition specificities (1). These enzymes represent the largest family of functionally related enzymes. R-M systems comprise pairs of intracellular enzymatic activities; that is, restriction endonuclease (REase) activity and DNA methyltransferase (MTase) activity. The REase recognizes specific sequences and catalyzes cleavage of double-stranded DNA. The MTase prevents the REase from digesting endogenous DNA by modifying the same target sequence with the methyl group from S-adenosyl-L-methionine (AdoMet) (2). R-M enzymes are classified into types I, II, III, and IV, based on their recognition sequence, subunit composition, cleavage position, and cofactor requirements (3). The simplest R-M systems are the type II enzymes. These generally consist of two separate enzymes, one responsible for restriction and the other for modification of the cognate DNA sequences. The orthodox type II REases are homodimers that typically recognize a palindromic sequence of 4 -8 bp and cut the DNA within or close to their recognition sequence in the presence of Mg 2ϩ to give a 5Ј-phosphate and a 3Ј-hydroxyl end. Although thousands of type II REases are known, the structures of only 19 have been reported to date (4). Structural and mutational analysis of several type II REases reveal that the sequence PDX n (D/E)XK (n ϭ 1-30 residues) acts as a catalytic/Mg 2ϩ binding signature motif (5). For the great majority of type II REases, Mg 2ϩ is an essential cofactor that can be substituted with Mn 2ϩ . The essential divalent ion cofactor is bound to the two carboxylates and the main carbon of the hydrophobic residue X of the PDX n (D/E)XK motif as well as to one of the non-bonding oxygens of the phosphate to be attacked. The fact that many REases have a common catalytic sequence motif suggests that they follow the same mechanism with at least one obvious exception, BfiI restriction endonuclease, which does not require a divalent metal ion cofactor (6).Type III R-M systems are multisubunit and multifunctional enzymes that exhibit bo...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A Mutation in the Mod Subunit of EcoP15I Restriction Enzyme Converts the DNA Methyltransferase to a Site-specific Endonuclease

Bist¹,

Madhusoodanan²,

Rao

2007

Journal of Biological Chemistry

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“…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).…”

mentioning

confidence: 99%

“…All other chemicals used were of the highest purity reagent grade. Sources for all other chemicals used in this study have been described earlier (6,20).…”

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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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Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis.

et al. 1995

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Type III restriction/modification systems recognize short non‐palindromic sequences, only one strand of which can be methylated. Replication of type III‐modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction. We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites. We have now addressed the molecular mechanism of site orientation‐specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation. The ATPase activity is uniquely recognition site‐specific, but EcoP15I‐modified sites also support the reaction. EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme‐to‐site ratio, in that site‐saturating enzyme levels elicit cleavage exclusively between the closest pair of head‐to‐head oriented sites. DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I‐DNA complexes.

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Interaction of EcoP15I DNA Methyltransferase with Oligonucleotides Containing the Asymmetric Sequence 5′-CAGCAG-3′

Cited by 39 publications

References 0 publications

A Mutation in the Mod Subunit of EcoP15I Restriction Enzyme Converts the DNA Methyltransferase to a Site-specific Endonuclease

A Mutation in the Mod Subunit of EcoP15I Restriction Enzyme Converts the DNA Methyltransferase to a Site-specific Endonuclease

Probing the Role of Cysteine Residues in the EcoP15I DNA Methyltransferase

Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis.

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