Investigation of the C-terminal domain of the bacterial DNA-(adenine N6)-methyltransferase CcrM

Maier, Johannes; Albu, Razvan F.; Jurkowski, Tomasz P.; Jeltsch, Albert

doi:10.1016/j.biochi.2015.10.011

Cited by 5 publications

(8 citation statements)

References 41 publications

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“…The mutants E280A and G305A display near-WT like K d’s on dsDNA. Our results for the mutants S315A, H317A, N330A and R350A differ with prior work on these mutants, reporting improved binding affinity over the WT enzyme using the same DNA and similar buffer conditions ( 28 , 29 ). Importantly, the prior work reported an extremely weak DNA affinity for the WT CcrM enzyme (2–10 μM compared to 75 nM shown in Supplementary Table S1 ), which may have resulted from using impure protein or only partially active enzyme ( 13 ).…”

Section: Resultscontrasting

confidence: 99%

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Cell cycle regulated DNA methyltransferase: fluorescent tracking of a DNA strand-separation mechanism and identification of the responsible protein motif

Konttinen

Carmody

Pathuri

et al. 2020

Nucleic Acids Research

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DNA adenine methylation by Caulobacter crescentus Cell Cycle Regulated Methyltransferase (CcrM) is an important epigenetic regulator of gene expression. The recent CcrM-DNA cocrystal structure shows the CcrM dimer disrupts four of the five base pairs of the (5′-GANTC-3′) recognition site. We developed a fluorescence-based assay by which Pyrrolo-dC tracks the strand separation event. Placement of Pyrrolo-dC within the DNA recognition site results in a fluorescence increase when CcrM binds. Non-cognate sequences display little to no fluorescence changes, showing that strand separation is a specificity determinant. Conserved residues in the C-terminal segment interact with the phospho-sugar backbone of the non-target strand. Replacement of these residues with alanine results in decreased methylation activity and changes in strand separation. The DNA recognition mechanism appears to occur with the Type II M.HinfI DNA methyltransferase and an ortholog of CcrM, BabI, but not with DNA methyltransferases that lack the conserved C-terminal segment. The C-terminal segment is found broadly in N4/N6-adenine DNA methyltransferases, some of which are human pathogens, across three Proteobacteria classes, three other phyla and in Thermoplasma acidophilum, an Archaea. This Pyrrolo-dC strand separation assay should be useful for the study of other enzymes which likely rely on a strand separation mechanism.

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Section: Resultscontrasting

confidence: 99%

“…We therefore purified the WT and all mutants using a C-terminal His-tag, resulting in high concentrations and purity ( Supplementary Figure S3 ). The purity of the proteins discussed here is significantly greater than previously published work which may contribute to the different results and conclusions ( 28 , 29 ).…”

Section: Resultscontrasting

confidence: 65%

Cell cycle regulated DNA methyltransferase: fluorescent tracking of a DNA strand-separation mechanism and identification of the responsible protein motif

Konttinen

Carmody

Pathuri

et al. 2020

Nucleic Acids Research

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“…Both SAND and PWWP domains were demonstrated to bind DNA nonspecifically 27,28 . Moreover, the C-terminal domain of CcrM was suggested to be involved in DNA binding 29 and deletion of the domain results in loss of enzymatic activity 15 . Indeed, in the current DNA-bound CcrM structure, molecule B provides almost all DNA phosphate contacts of the non-target strand, with interactions mediated by the C-terminal domain concentrated on the four 5’ phosphates surrounding the recognition sequence (Figs.…”

Section: Resultsmentioning

confidence: 99%

The cell cycle-regulated DNA adenine methyltransferase CcrM opens a bubble at its DNA recognition site

et al. 2019

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The Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM) methylates the adenine of hemimethylated GANTC after replication. Here we present the structure of CcrM in complex with double-stranded DNA containing the recognition sequence. CcrM contains an N-terminal methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the 5-base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson–Crick polar hydrogen bonds to ensure high enzymatic specificity.

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“…4a and b ). The methylated DNA substrate was prepared by in vitro DNA methylation of the unmethylated substrate with purified His 6 -tagged CcrM 46 . Methylation of the PCR product (60 ng μl −1 ) was performed in reaction buffer (50 mM HEPES pH 7.0, 50 mM NaCl, 1 mM EDTA, 0.5 mM DTT, and 5 μg ml −1 BSA) supplemented with 200 μM S-adenosyl- L -methionine (Sigma) and 5 μM CcrM for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Design of synthetic epigenetic circuits featuring memory effects and reversible switching based on DNA methylation

2017

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Epigenetic systems store information in DNA methylation patterns in a durable but reversible manner, but have not been regularly used in synthetic biology. Here, we designed synthetic epigenetic memory systems using DNA methylation sensitive engineered zinc finger proteins to repress a memory operon comprising the CcrM methyltransferase and a reporter. Triggering by heat, nutrients, ultraviolet irradiation or DNA damaging compounds induces CcrM expression and DNA methylation. In the induced on-state, methylation in the operator of the memory operon prevents zinc finger protein binding leading to positive feedback and permanent activation. Using an mf-Lon protease degradable CcrM variant enables reversible switching. Epigenetic memory systems have numerous potential applications in synthetic biology, including life biosensors, death switches or induction systems for industrial protein production. The large variety of bacterial DNA methyltransferases potentially allows for massive multiplexing of signal storage and logical operations depending on more than one input signal.

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Investigation of the C-terminal domain of the bacterial DNA-(adenine N6)-methyltransferase CcrM

Cited by 5 publications

References 41 publications

Cell cycle regulated DNA methyltransferase: fluorescent tracking of a DNA strand-separation mechanism and identification of the responsible protein motif

Cell cycle regulated DNA methyltransferase: fluorescent tracking of a DNA strand-separation mechanism and identification of the responsible protein motif

The cell cycle-regulated DNA adenine methyltransferase CcrM opens a bubble at its DNA recognition site

Design of synthetic epigenetic circuits featuring memory effects and reversible switching based on DNA methylation

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