A base-flipping phenomenon has been established for DNA methyltransferases and for DNA base-excision repair glycosylases and is likely to prove general for enzymes that need access to DNA bases to undergo chemical reaction. T4 phage beta-glucosyltransferase (BGT) is a good candidate for this novel mechanism. In order to confirm this, BGT was crystallized with an abasic site-containing DNA and uridine diphosphoglucose (UDP-glucose). The crystallization strategy is described. A complete data set was collected at 1.8 A resolution on a Cu Kalpha rotating-anode X-ray source. Molecular replacement was performed and the initial electron-density maps clearly show bound DNA.
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http://dx.doi.org/10.1107/S0907444902012969 | DOI Listing |
Biosens Bioelectron
October 2023
College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China; School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China. Electronic address:
β-glucosyltransferase (β-GT) can specifically catalyze the conversion of 5-hydroxymethylcytosine (5-hmC) to 5-glucosylhydroxy methylcytosine (5-ghmC), and it is associated with the control of phage-specific gene expression by affecting transcription process in vivo and in vitro. The current strategies for β-GT assay usually involve expensive equipment, laborious treatment, radioactive hazard, and poor sensitivity. Here, we report a Spinach-based fluorescent light-up biosensor for label-free measurement of β-GT activity by utilizing 5-hmC glucosylation-initiated rolling circle transcription amplification (RCTA).
View Article and Find Full Text PDFAnal Chem
October 2021
School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China.
Methods Mol Biol
March 2021
New England Biolabs, Inc., Ipswich, MA, USA.
The oxidation activity of the mammalian ten-eleven translocation dioxygenase (TET) on 5-methylcytosine (5mC) of DNA is usually monitored by analytical methods such as dot blotting and liquid chromatography-mass spectrometry (LC-MS). Herein, we describe a high throughput capillary gel electrophoresis assay for monitoring the in vitro oxidation of 5mC by TET. The method is rapid and quantitative, and can serve as a powerful tool in mechanistic studies of TET.
View Article and Find Full Text PDFAnal Chem
April 2016
Division of Physical Biology and Bioimaging Centre, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences , Shanghai 201800, China.
DNA hydroxymethylation (5-hmC) is a kind of new epigenetic modification, which plays key roles in DNA demethylation, genomic reprogramming, and the gene expression in mammals. For further exploring the functions of 5-hmC, it is necessary to develop sensitive and selective methods for detecting 5-hmC. Herein, we developed a novel multiplexing electrochemical (MEC) biosensor for 5-hmC detection based on the glycosylation modification of 5-hmC and enzymatic signal amplification.
View Article and Find Full Text PDFBiosens Bioelectron
May 2016
Institute of Analytical Science, Northwest University, Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Xi'an, Shaanxi 710069, China.
An electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive discrimination of DNA hydroxymethylation and assay of the β-glucosyltransferase (β-GT) activity was developed. The ECL biosensing electrode was fabricated by gold nanoparticles (AuNPs)/Nafion film, and then, tris(2, 2'-ripyridine) dichlororuthenium(II) (Ru(bpy)3(2+)) was electrostatically adsorbed into the AuNPs/Nafion film, finally, the hydroxymethylated double-stranded DNA (ds-DNA)-tagged with ferrocene was self-assembled onto the surface of the AuNPs. When β-GT and uridine diphosphoglucose (UDP-Glu) were introduced, the hydroxymethylcytosine residues within 5'-CCGG-3' of ds-DNA on the biosensing electrode were glucosylated.
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