Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence

Reddy, Yeturu V. R.; Rao, Desirazu N.

doi:10.1006/jmbi.2000.3673

Cited by 51 publications

(33 citation statements)

References 45 publications

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“…The fluorescence of 2AP when substituted for the methylatable base is often, but not always, an effective probe of this base flipping mechanism as the fluorescence is heavily quenched in a DNA duplex but the quenching is greatly reduced in the catalytic site of a base flipping enzyme (34,39–41). The equilibrium position of the 2AP is heavily weighted towards a base-paired, stacked, low-fluorescence location within the DNA double helix in the absence of a methyltransferase, and the equilibrium is shifted towards a base-flipped highly fluorescent position in the presence of the methyltransferase.…”

Section: Resultsmentioning

confidence: 99%

DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping

Su¹

2005

Nucleic Acids Research

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The maintenance methyltransferase M.EcoKI recognizes the bipartite DNA sequence 5 0 -AACNNN-NNNGTGC-3 0 , where N is any nucleotide. M.EcoKI preferentially methylates a sequence already containing a methylated adenine at or complementary to the underlined bases in the sequence. We find that the introduction of a single-stranded gap in the middle of the non-specific spacer, of up to 4 nt in length, does not reduce the binding affinity of M.EcoKI despite the removal of non-sequencespecific contacts between the protein and the DNA phosphate backbone. Surprisingly, binding affinity is enhanced in a manner predicted by simple polymer models of DNA flexibility. However, the activity of the enzyme declines to zero once the single-stranded region reaches 4 nt in length. This indicates that the recognition of methylation of the DNA is communicated between the two methylation targets not only through the protein structure but also through the DNA structure. Furthermore, methylation recognition requires base flipping in which the bases targeted for methylation are swung out of the DNA helix into the enzyme. By using 2-aminopurine fluorescence as the base flipping probe we find that, although flipping occurs for the intact duplex, no flipping is observed upon introduction of a gap. Our data and polymer model indicate that M.EcoKI bends the non-specific spacer and that the energy stored in a doublestranded bend is utilized to force or flip out the bases. This energy is not stored in gapped duplexes. In this way, M.EcoKI can determine the methylation status of two adenine bases separated by a considerable distance in double-stranded DNA and select the required enzymatic response.

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Section: Resultsmentioning

confidence: 99%

DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping

Su¹

2005

Nucleic Acids Research

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“…19,21,22,25,26 However, the effect is not always only due to base flipping and occasionally it does not occur at all such that controls are necessary to demonstrate the causal relationship of base flipping and fluorescence increase. 27,28 Adenine-N6 MTases are characterized by a conserved (D/N/S)PP(Y/F) motif that forms the active site of the enzyme 29,30 (Figure 1A). Structural and biochemical studies have provided evidence that the D/N/S residue contacts the exocyclic amino group of the flipped base and activates it for methyl group transfer 15,20,30,31 and that the aromatic amino acid residue stacks to the flipped target base and stabilizes the flipped conformation.…”

Section: Introductionmentioning

confidence: 99%

Stopped-flow and Mutational Analysis of Base Flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase

Liebert¹,

Hermann²,

Schlickenrieder³

et al. 2004

Journal of Molecular Biology

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“…This method takes advantage of the fact that 2AP fluorescence quantum yield increases when its environment becomes more polar (12). MTases that enhance 2AP's fluorescence when the fluorophore replaces the base that is normally methylated include M.EcoRI (13), M.TaqI (14), M.HhaI (14), M.EcoP151 (15), M.EcoKI (16), M.RsrI (17), T4 Dam (18), M.KpnI (19) and E. coli Dam (20). However, some exceptions to this general rule also exist.…”

Section: Introductionmentioning

confidence: 99%

“…This includes M.EcoRV, which did not enhance 2AP fluorescence when it substituted the target base for methylation (21). Additionally, MTases M.EcoKI (16), M.EcoRV (21) and M.EcoP151 (15) enhanced 2APs fluorescence when it was placed at positions other than the base to which the methyl group is transferred.…”

Section: Introductionmentioning

confidence: 99%

DNA base flipping by both members of the PspGI restriction-modification system

Carpenter

Bhagwat

2008

Nucleic Acids Research

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The PspGI restriction–modification system recognizes the sequence CCWGG. R.PspGI cuts DNA before the first C in the cognate sequence and M.PspGI is thought to methylate N4 of one of the cytosines in the sequence. M.PspGI enhances fluorescence of 2-aminopurine in DNA if it replaces the second C in the sequence, while R.PspGI enhances fluorescence when the fluorophore replaces adenine in the central base pair. This strongly suggests that the methyltransferase flips the second C in the recognition sequence, while the endonuclease flips both bases in the central base pair out of the duplex. M.PspGI is the first N4-cytosine MTase for which biochemical evidence for base flipping has been presented. It is also the first type IIP methyltransferase whose catalytic activity is strongly stimulated by divalent metal ions. However, divalent metal ions are not required for its base-flipping activity. In contrast, these ions are required for both base flipping and catalysis by the endonuclease. The two enzymes have similar temperature profiles for base flipping and optimal flipping occurs at temperatures substantially below the growth temperature of the source organism for PspGI and for the catalytic activity of endonuclease. We discuss the implications of these results for DNA binding by these enzymes and their evolutionary origin.

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Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence

Cited by 51 publications

References 45 publications

DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping

DNA bending by M.EcoKI methyltransferase is coupled to nucleotide flipping

Stopped-flow and Mutational Analysis of Base Flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase

DNA base flipping by both members of the PspGI restriction-modification system

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