Presteady state kinetics of an S-adenosylmethionine-dependent enzyme. Evidence for a unique binding orientation requirement for EcoRI DNA methyltransferase

Reich, Norbert O.; Mashhoon, Neda

doi:10.1016/s0021-9258(18)98334-1

Cited by 50 publications

(26 citation statements)

References 11 publications

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“…15,16 CcrM shows a surprising level of sequence discrimination (Figure 2C and Table 1E, dsDNA). 18,19 Single-base pair changes in the recognition sequence [e.g., 5′-GACTC-3′ to 5′-AACTC-3′, which does not include the target A (italicized)] result in ≤10 6 -fold decreases in the apparent k methylation and a 1.9 × 10 7 -fold change in specificity (k methylation /K d ). This level of sequence discrimination is at least 10 3 −10 4 -fold greater than what is typically observed with other DNA MTases.…”

Section: ■ Resultsmentioning

confidence: 99%

“…Direct measurement of the methylation constant for dsDNA (Figure C; k methylation , 0.62 ± 0.08 min –1 ) confirms that methylation or a conformational step prior to methylation limits the turnover of the enzyme. The k methylation constant is particularly slow when compared to those of other DNA adenine N 6 MTases such as EcoRI (2460 min –1 ) and T4 Dam (36 min –1 ). , CcrM shows a surprising level of sequence discrimination (Figure C and Table E, dsDNA). , Single-base pair changes in the recognition sequence [e.g., 5′-G A CTC-3′ to 5′- A A CTC-3′, which does not include the target A (italicized)] result in ≤10 6 -fold decreases in the apparent k methylation and a 1.9 × 10 7 -fold change in specificity ( k methylation / K d ). This level of sequence discrimination is at least 10 3 –10 4 -fold greater than what is typically observed with other DNA MTases …”

Section: Resultsmentioning

confidence: 99%

“…Adenine and cytosine DNA MTases uniformly show rate-limiting product release, with the methyl transfer and associated conformational changes occurring 40–300-fold faster, producing a well-known burst in product formation in steady state plots. ,− Surprisingly, CcrM does not display a classic “burst” of product formation (Figure D). Moreover, single-turnover experiments (Figure C and Table A–D) with dsDNA show that k methylation or the rate of a prior first-order step ranges from 0.62 to 8.09 min –1 , depending on the variable “ N ” and the flanking sequence.…”

Section: Discussionmentioning

confidence: 99%

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Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition

2017

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Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5'-GANTC-3' in single-stranded and hemimethylated double-stranded DNA. We report the K, k, k, and K for single-stranded and hemimethylated substrates, revealing discrimination of 10-fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

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Section: ■ Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition

2017

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“…It is thus expected that the methylation of each of the strands will proceed from a different unique binding orientation. Support for this postulate has been demonstrated for the EcoRl methylase using a pre-steady-state kinetic analysis (Reich & Mashhoon, 1993). Only one methylase-DNA crystal structure has been reported, that for Hhal (Klimasauskas et al, 1994), which shows that the enzyme makes contacts to the target bases and phosphates of both strands.…”

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confidence: 97%

Probing the Protein-DNA Interface of the EcoRV Modification Methyltransferase Bound to Its Recognition Sequence, GATATC

Szczelkun¹,

Jones²,

Connolly³

1995

Biochemistry

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The DNA contacts produced between the EcoRV modification methyltransferase and its recognition sequence, GATATC, have been determined. The enzyme's general location in a methylase/DNA/sinefungin ternary complex was evaluated by protection from exonuclease III digestion. Important phosphate contacts were resolved using N-ethyl-N-nitrosourea ethylation interference footprinting. Methylation protection and interference using dimethyl sulfate were employed to assess significant contacts to purinic bases. The protein-DNA interface was further probed using oligodeoxynucleotides containing base analogues within the GATATC sequence. Most of the experiments were carried out using hemimethylated sequences, i.e., having 6-methyladenosine at the methylation site in one of the strands. The monomeric methylase was found to bind to the DNA in two different orientations for the methylation of each strand. The enzyme approaches the DNA, predominantly from one "side", and makes most of its contacts in the major groove. In either of the two binding events contacts are made to the four phosphates NpNpNpGpA and the three bases GAT (where GAT represents the 5' half of the GATATC site) on both DNA strands. The phosphates and bases in the 3' ATC half are much less important. Although the contacts made to the equivalent locations on each strand are similar, they display a slight but consistent change dependent on which strand contains the 6-methyldeoxyadenosine. This strand variation shows completely reciprocal behavior, switching around exactly, depending entirely on the methylated deoxyadenosine location. It is this that provides evidence for the two binding modes. The results obtained are discussed in terms of possible models for the protein-DNA interface.

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“…The MTase is highly efficient, with a specificity constant (kat/Km) for plasmid DNA of greater than 108 s~* M-1, and a large component of the extreme efficiency derives from a facilitated diffusion mechanism.2 The enzyme catalyzes methylation of one strand of duplex DNA and dissociates from the DNA prior to any subsequent catalytic events2 (Rubin & Modrich, 1977;Reich & Mashhoon, 1991). Furthermore, only one strand can be methylated from a single binding orientation (Reich & Mashhoon, 1993). The £coRI MTase reaction occurs by direct attack of the N6 nitrogen onto the methylsulfonium and not via the attack by the more nucleophilic N1 center in the purine ring (Santi et al, 1988) (see Figure 1).…”

mentioning

confidence: 99%

Investigation of Ionizable Residues Critical for Sequence-Specific Enzymic DNA Modification: Protein Modification and Steady-State and Pre-Steady-State Kinetic pH Analyses of EcoRI DNA Methyltransferase

Mashhoon

Reich²

1994

Biochemistry

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Steady-and pre-steady-state pH kinetic analyses are widely used methods to investigate important ionizable groups in enzyme-catalyzed reactions. The first such analysis to identify ionizable residues critical for sequence-specific modification of DNA is presented. EcoRI DNA methyltransferase uses S-adenosyl-L-methionine (AdoMet) to catalyze the N6 methylation of the second adenine in the double-stranded DNA sequence GAATTC. The kinetic mechanism was previously shown to be steady-state-ordered bi bi in which AdoMet binds first followed by DNA addition [Reich, N. O., & Mashhoon, N. (1991) Biochemistry 30, 2933-2939]. Steady-state parameters are strongly dependent on pH and implicate at least four residues with pKa values between 8.2 and 8.9 in the free enzyme and AdoMet-Bound enzyme and one residue with an apparent pKa of 6.0. The data obtained are consistent with the enzyme binding the form of AdoMet in which the alpha amino group is protonated. Two protein residues with an apparent pKa between 8.9 and 9.2 were implicated within the central complex (enzyme-DNA-AdoMet). The general insensitivity of all steady-state parameters to pH changes between pH 6.0 and 8.0 suggests that no critical protein residues undergo ionization-state changes in this range. The lack of significant pH-dependent changes in protein fluorescence and DNA thermal stability suggests minimal structural changes in either macromolecule. In support of the steady-state results single-turnover experiments reveal minimal pH dependence of the methylation rate constant between pH 5.53 and 8.6. Thus, no amino acids critical for catalysis undergo ionization-state changes in this range.(ABSTRACT TRUNCATED AT 250 WORDS)

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Presteady state kinetics of an S-adenosylmethionine-dependent enzyme. Evidence for a unique binding orientation requirement for EcoRI DNA methyltransferase

Cited by 50 publications

References 11 publications

Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition

Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition

Probing the Protein-DNA Interface of the EcoRV Modification Methyltransferase Bound to Its Recognition Sequence, GATATC

Investigation of Ionizable Residues Critical for Sequence-Specific Enzymic DNA Modification: Protein Modification and Steady-State and Pre-Steady-State Kinetic pH Analyses of EcoRI DNA Methyltransferase

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