Deinococcus radiodurans DNA transmethylase

A technology of Deinococcus radiodurans and methyltransferase, applied in transferase, enzyme, enzyme and other directions, can solve the problems of M.DraR1 modification mode and substrate recognition sequence that have not been reported.

Pending Publication Date: 2019-03-08
ZHEJIANG UNIV
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

which body M.DraR1 gene (named dr_C0020 , the protein sequence ID is ANC73351.1), the expressed product DNA methyltransferase M.DraR1 consists of 434 amino acids, has a typical DNA methylt

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Deinococcus radiodurans DNA transmethylase
  • Deinococcus radiodurans DNA transmethylase
  • Deinococcus radiodurans DNA transmethylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Deinococcus radiodurans DNA methyltransferase M.DraR1 substrate recognition specificity

[0020] (1) DNA methyltransferase M.DraR1 Construction of gene deletion strains: using the classic "three-segment linking method" to M.DraR1 The upstream fragment (about 710 bp) and downstream fragment (about 1000 bp) of the gene sequence and the resistance gene fragment (streptomycin resistance gene Str, about 927 bp) are linked into a whole fragment in vitro by T4 ligase (Takara company) (See figure 1 A), and then transform this triple product into competent cells of Deinococcus radiodurans, screen the mutant strains on the Str-resistant TGY medium plate, and obtain the result by PCR and sequencing verification M.DraR1 Gene deletion strain—Δ M.DraR1 (See figure 1 B).

[0021] (2) Deinococcus radiodurans wild-type DraR1 and M.DraR1 Extraction of gene knockout strain genome: pick wild-type DraR1 and knockout strain Δ M.DraR1 Inoculate a single colony into 5 mL of...

Embodiment 2

[0027] Example 2: In vivo enzyme activity analysis of DNA methyltransferase M.DraR1

[0028] (1) Construction of the in vivo expression vector pRRS-M.DraR1 vector: using the genomic DNA of Deinococcus radiodurans as a template, using pfu High-fidelity polymerase (full gene, AP 221) was used to amplify the M.DraR1 gene in vitro by PCR. The upstream primer pRRS-M.DraR1-F contained the Sbf I restriction site, the TTAAGG box and the TTAATCAT sequence. The sequence is shown in SEQ ID NO. 2; the downstream primer pRRS-M.DraR1-R contains a BamH I restriction site and a CCGCGG substrate conservative sequence, and its sequence is shown in SEQ ID NO. 3. 1.0% agarose electrophoresis detection, purification of PCR products (Life company DNA purification kit, Cat No. 116401), purified PCR fragments and pRRS plasmid (gifted by Professor Roberts of NEB company) and Sbf I and BamH I (NEB company) at the same time Carry out enzyme digestion, purify the digested fragments, connect them with T...

Embodiment 3

[0032] Example 3: Analysis of DNA methyltransferase M.DraR1 in vitro enzyme activity

[0033] (1) Linearization treatment of pRRS-M.DraR1 plasmid: Cut the above obtained plasmid into linear fragments with Hind III, purify and recover for later use.

[0034] (2) DNA methyltransferase M.DraR1 in vitro reaction system: Add 1-2 μg of the above-mentioned treated plasmid into a solution containing 50-200 mM KCl, 10-50 mM Tris-HCl (pH7.5-8.0), 0.1 mM Add 1 μM purified M.DraR1 protein to the reaction buffer of EDTA, 3-7 mM β-Me and 20-100 μM SAM, and react under suitable conditions.

[0035] (3) Optimum temperature range for the enzyme activity of DNA methyltransferase M.DraR1: place the above reaction systems at 4-60 °C for 0.5-1 h, and purify the plasmid DNA or phage DNA after the reaction. The results further indicated that M.DraR1 methyltransferase can methylate and modify the "CCGCGG" conservative motif, and its optimum temperature range is 25-37 ℃. At 4 °C, it also has weak me...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses deinococcus radiodurans DNA transmethylase. The DNA transmethylase is derived from deinococcus radiodurans, and has typical DNA transmethylase conserved structural domains which are an Adomet binding domain containing a 'FxGxG' conserved sequence, a target sequence recognition domain (a TRD sequence) and a catalytic domain containing a 'TSPPY' conserved sequence successively from a N-terminal to a C-terminal, and belongs to Alpha-type N4-Cytosine DNA transmethylase. A substrate DNA conserved sequence recognized by the DNA transmethylase is 5'-CCGCGG-3', a position of methylated modification is a N4 site of a second cytosine C, and 4 mC type of a modified base is generated. An optimum temperature of a methylation reaction thereof is between 25 and 37 DEG C. The related DNA transmethylase is capable of specific-recognizing a 'CCGCGG' conserved sequence motif and methylated-modifying the N4 site of the second cytosine C thereof, and generating the 4mC modified base, and is the N4-Cytosine DNA transmethylase.

Description

technical field [0001] The invention relates to a Deinococcus radiodurans DNA methyltransferase M.DraR1, and relates to the DNA conservative sequence features recognized by it and the methylation modification mode thereof. Background technique [0002] DNA modification can cause changes in chromatin structure, DNA conformation and stability, leading to changes in the interaction between DNA and proteins, regulating gene expression and expanding the structural complexity and information depth of DNA. The apparent information conveyed by the chemical modification of four bases exists in all living organisms including viruses and phages. DNA methylation modification is a widespread and very important DNA epigenetic modification phenomenon, which plays an important regulatory role in the growth, maintenance of genome stability and differentiation of organisms. [0003] The enzymes that mediate and catalyze DNA methylation modification are mainly DNA methyltransferases, which ar...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N9/10
CPCC12N9/1007
Inventor 华跃进李胜杰王梁燕蔡建玲华孝廷
Owner ZHEJIANG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap