Molecular Drivers of Base Flipping During Sequence‐Specific DNA Methylation

Matje, Douglas M.; Reich, Norbert O.

doi:10.1002/cbic.201200104

Cited by 5 publications

(14 citation statements)

References 41 publications

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“…Thus, we suggest that the loop does not act to directly eject the base from the helix but instead closes once the base has begun to vacate the DNA helix. Our recent work using miscognate sites 23 (sites with a single-base pair substitution) suggests that upon location of the target sequence, compression of the protein domains around the DNA destabilizes the target base pair, in agreement with several molecular dynamics models of the flipping process. 2,19,20 The observed decrease in the flipping rate with the W41F mutant validates the computational predictions that cofactor binding facilitates compression of the domains to destabilize the target cytosine.…”

Section: ■ Materials and Methodssupporting

confidence: 80%

Distal Structural Elements Coordinate a Conserved Base Flipping Network

et al. 2013

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One of the most dramatic illustrations of enzymatic promotion of a high-energy intermediate is observed in DNA modification and repair enzymes where an individual base is rotated (flipped) 180° around the deoxyribose-phosphate backbone and into the active site. While the end states have been extensively characterized, experimental techniques have yet to yield a full description of the base flipping process and the role played by the enzyme. The C5 cytosine methyltransferase M.HhaI coordinates an ensemble of reciprocal DNA and enzyme rearrangements to efficiently flip the target cytosine from the DNA helix. We sought to understand the role of individual amino acids during base flipping. Our results demonstrate that M.HhaI initiates base flipping before closure of the catalytic loop and utilizes the conserved serine 85 in the catalytic loop to accelerate flipping and maintain distortion of the DNA backbone. Serine 87, which forms specific contacts within the DNA helix after base flipping, is not involved in the flipping process or in maintaining the catalytically competent complex. At the base of the catalytic loop, glycine 98 acts as a hinge to allow conformational dynamism of the loop and mutation to alanine inhibits stabilization of the closed loop. Our results illustrate how an enzyme utilizes numerous, distal residues in concert to transform substrate recognition into catalysis.

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Section: ■ Materials and Methodssupporting

confidence: 80%

Distal Structural Elements Coordinate a Conserved Base Flipping Network

et al. 2013

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“…The decay parameters reported here are quantitatively consistent with previously reported fluorescence intensity measurements. 14,19,20 Moreover, the E94W and E94W/W41F mutants are observed to have essentially identical decay signals when the enzyme is saturated with cofactor (see sTable 2 of the Supporting Information). This demonstrates that the fluorescence of the native tryptophan is efficiently quenched by cofactor binding and signal changes in the ternary complexes of the E94W mutant are solely from the tryptophan engineered into the loop.…”

Section: ■ Resultsmentioning

confidence: 99%

“…Timeresolved measurements were taken on an Applied Photophysics SX.18MV apparatus as described previously. 14,20,21 NMR Spectroscopy. All experiments were conducted at 25 °C either on a Bruker 800 MHz UltraShield Plus II NMR spectrometer equipped with a four-channel ( 1 H/ 13 .…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Enzyme-Promoted Base Flipping Controls DNA Methylation Fidelity

et al. 2013

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A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme-substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.HhaI has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein-DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.

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“…Research shows that closure of the catalytic loop occurs after base flipping [9, 58] and that the enzyme utilizes the hydrogen bonds between Gln237 and Ser87 to lock the flipped base. Other research studies indicate that target base flipping and closure of the mobile catalytic loop occur simultaneously [17].…”

Section: Resultsmentioning

confidence: 99%

Metadynamics Simulation Study on the Conformational Transformation of HhaI Methyltransferase: An Induced-Fit Base-Flipping Hypothesis

Jin

Zhao

et al. 2014

BioMed Research International

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DNA methyltransferases play crucial roles in establishing and maintenance of DNA methylation, which is an important epigenetic mark. Flipping the target cytosine out of the DNA helical stack and into the active site of protein provides DNA methyltransferases with an opportunity to access and modify the genetic information hidden in DNA. To investigate the conversion process of base flipping in the HhaI methyltransferase (M.HhaI), we performed different molecular simulation approaches on M.HhaI-DNA-S-adenosylhomocysteine ternary complex. The results demonstrate that the nonspecific binding of DNA to M.HhaI is initially induced by electrostatic interactions. Differences in chemical environment between the major and minor grooves determine the orientation of DNA. Gln237 at the target recognition loop recognizes the GCGC base pair from the major groove side by hydrogen bonds. In addition, catalytic loop motion is a key factor during this process. Our study indicates that base flipping is likely to be an “induced-fit” process. This study provides a solid foundation for future studies on the discovery and development of mechanism-based DNA methyltransferases regulators.

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Molecular Drivers of Base Flipping During Sequence‐Specific DNA Methylation

Cited by 5 publications

References 41 publications

Distal Structural Elements Coordinate a Conserved Base Flipping Network

Distal Structural Elements Coordinate a Conserved Base Flipping Network

Enzyme-Promoted Base Flipping Controls DNA Methylation Fidelity

Metadynamics Simulation Study on the Conformational Transformation of HhaI Methyltransferase: An Induced-Fit Base-Flipping Hypothesis

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