Type III DNA restriction and modification systems EcoP1 and EcoP15

Hümbelin, Markus; Suri, B.; Rao, Desirazu N.; Hornby, D.; Eberle, Helen; Pripfl, Theres; Kenel, Susanne; Bickle, Thomas A.

doi:10.1016/0022-2836(88)90330-0

Cited by 93 publications

(36 citation statements)

References 32 publications

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“…In the presence of 6 M guanidinium chloride, the remaining two cysteines react with DTNB (Table I). These results clearly indicate the possible absence of disulfides in the enzyme and also corroborate the amino acid sequence data of the protein which shows the presence of six cysteine residues (28). To examine whether disulfide bonds were present in M. EcoP15I and, if present, were important for activity, we tested the effects of DTT, a strong disulfide bond reducing agent.…”

Section: Effect Of Thiol Reagents On Ecop15i Dna Methyltransferasesupporting

confidence: 49%

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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Section: Effect Of Thiol Reagents On Ecop15i Dna Methyltransferasesupporting

confidence: 49%

Probing the Role of Cysteine Residues in the EcoP15I DNA Methyltransferase

Reddy

Rao

1998

Journal of Biological Chemistry

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“…Southern (31,44), it is conceivable that the species specificity of the DNA region is due to its location on a prophage the host range of which is restricted to C. glutamicum strains. It is known that prophages can carry genes for modification and restriction enzymes (20). Gene disruption experiments showed that inactivation of orfl resulted in a dramatic increase of fertility, whereas disruption of orf2 only slightly raised fertility.…”

Section: K S R F I E Q G a L E Y G E D A T Q E N I D R F G R T H Mmentioning

confidence: 99%

Cloning and characterization of a DNA region encoding a stress-sensitive restriction system from Corynebacterium glutamicum ATCC 13032 and analysis of its role in intergeneric conjugation with Escherichia coli

et al. 1994

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RP4-mediated transfer of mobilizable plasmids in intergeneric conjugation of Escherichia coli donors with Corynebacterium glutamicum ATCC 13032 is severely affected by a restriction system in the recipient that can be inactivated by a variety of exogenous stress factors. In this study a rapid test procedure based on intergeneric conjugal plasmid transfer that permitted the distinction between restriction-negative and restriction-positive C. gludamicum clones was developed. By using this procedure, clones of the restrictiondeficient mutant strain C. glutamicum RM3 harboring a plasmid library of the wild-type chromosome were checked for their restriction properties. A complemented clone with a restriction-positive phenotype was isolated and found to contain a plasmid with a 7-kb insertion originating from the wild-type chromosome. This plasmid, termed pRES806, is able to complement the restriction-deficient phenotype of different C. glutamicum mutants. Sequence analysis revealed the presence of two open reading frames (orfl and orfj) on the complementing DNA fragment. The region comprising orfi and orf2 displayed a strikingly low G+C content and was present exclusively in C. glutamicum strains. Gene disruption experiments with the wild type proved that orfi is essential for complementation, but inactivation of o42 also resulted in a small but significant increase in fertility. These results were confirmed by infection assays with the bacteriophage CL31 from Corynebacterum lilum ATCC 15990.

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“…EcoP15I DNA methyltransferase (M.EcoP15I) adds a methyl group to the second adenine in the recognition sequence 5Ј-CAGCAG-3Ј in the presence of AdoMet (6). It is an N 6 -adenine MTase, and like all N 6 -adenine MTases, M.EcoP15I contains two highly conserved sequences, FXGXG (motif I) at position 444 -448 and DPPY (motif IV) at position 125-128 (7). Although mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered, mutations in motif IV resulted in loss of enzyme activity, but AdoMet and DNA binding were not affected (8).…”

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confidence: 99%

“…An inspection of amino acid sequences of DNA methyltransferases did not reveal any characteristic metal binding motif. However, in EcoP15I DNA methyltransferase, a PD(X) n (D/E)XK-like motif is present in which the partially conserved proline is replaced by methionine (7).…”

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Identification and Mutational Analysis of Mg2+ Binding Site in EcoP15I DNA Methyltransferase

Bist¹,

Rao

2003

Journal of Biological Chemistry

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EcoP15I DNA methyltransferase catalyzes the transfer of the methyl group of S-adenosyl-L-methionine to the N 6 position of the second adenine within the doublestranded DNA sequence 5-CAGCAG-3. To achieve catalysis, the enzyme requires a magnesium ion. Binding of magnesium to the enzyme induces significant conformational changes as monitored by circular dichroism spectroscopy. EcoP15I DNA methyltransferase was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at pH 8.0. The inactivated enzyme was cleaved into two fragments with molecular masses of 36 and 35 kDa. Using this affinity cleavage assay, we have located the magnesium bindinglike motif to amino acids 355-377 of EcoP15I DNA methyltransferase. Sequence homology comparisons between EcoP15I DNA methyltransferase and other restriction endonucleases allowed us to identify a PD(X) n (D/E)XK-like sequence as the putative magnesium ion binding site. Point mutations generated in this region were analyzed for their role in methyltransferase activity, metal coordination, and substrate binding. Although the mutant methyltransferases bind DNA and S-adenosyl-L-methionine as well as the wild-type enzyme does, they are inactive primarily because of their inability to flip the target base. Collectively, these data are consistent with the fact that acidic amino acid residues of the region 355-377 in EcoP15I DNA methyltransferase are important for the critical positioning of magnesium ions for catalysis. This is the first example of metal-dependent function of a DNA methyltransferase. These findings provide impetus for exploring the role(s) of metal ions in the structure and function of DNA methyltransferases. DNA methyltransferases (MTases)1 sequence-specifically modify DNA in a wide range of organisms. The enzymes require the methyl donor S-adenosyl-L-methionine (AdoMet) to modify their target base (1). Three classes of DNA MTases, namely, the C 5 -cytosine MTases, N 4 -cytosine MTases, and N 6 -adenine-specific MTases have been identified on the basis of sequence homology and type of methylation catalyzed (2, 3). Structure-guided sequence comparison analysis revealed that all three classes of MTase are closely related to one another (4, 5). EcoP15I DNA methyltransferase (M.EcoP15I) adds a methyl group to the second adenine in the recognition sequence 5Ј-CAGCAG-3Ј in the presence of AdoMet (6). It is an N 6 -adenine MTase, and like all N 6 -adenine MTases, M.EcoP15I contains two highly conserved sequences, FXGXG (motif I) at position 444 -448 and DPPY (motif IV) at position 125-128 (7). Although mutations in motif I completely abolished AdoMet binding but left target DNA recognition unaltered, mutations in motif IV resulted in loss of enzyme activity, but AdoMet and DNA binding were not affected (8). By employing chemical modification using thiol-directed agents and site-directed mutagenesis, it was demonstrated that cysteine 344 in M.EcoP15I was necessary for enzyme activity and played an essential role in DNA binding (9). W...

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Type III DNA restriction and modification systems EcoP1 and EcoP15

Cited by 93 publications

References 32 publications

Probing the Role of Cysteine Residues in the EcoP15I DNA Methyltransferase

Probing the Role of Cysteine Residues in the EcoP15I DNA Methyltransferase

Cloning and characterization of a DNA region encoding a stress-sensitive restriction system from Corynebacterium glutamicum ATCC 13032 and analysis of its role in intergeneric conjugation with Escherichia coli

Identification and Mutational Analysis of Mg2+ Binding Site in EcoP15I DNA Methyltransferase

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