Cloning and analysis of biochemical and catalytic properties of DNA methyltransferase M1.BspACI

M. V. Tarasova; V. V. Kuznetsov; N. A. Netesova; D. A. Gonchar; S. Kh Degtyarev

doi:10.3103/S0096392511020106

Cloning and analysis of biochemical and catalytic properties of DNA methyltransferase M1.BspACI

M. V. Tarasova, V. V. Kuznetsov, N. A. Netesova, D. A. Gonchar, S. Kh Degtyarev

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Abstract

DNA methyltransferases genes of the BspACI restriction-modification system from Bacillus psychrodurans AC have been cloned in E. coli cells. Analysis of amino acid sequences of the proteins showed that both of these genes belong to C5 DNA methyltransferases. Gene M1. BspACI has been subcloned in pJW2 vector. A high-purity recombinant enzyme has been obtained using chromatography on different carriers. It has been shown that M1. BspACI modifies the first cytosine residue in the sequence 5′-CCGC-3′. Kinetic parameters of DNA methylation by the enzyme have been determined. Catalytic constant appears to be 0.095 ± 0.002 min^-1. K_{m phage is λ DNA}-0.053 ± 0.007 μM, and K_mSAM is 5.1 ± 0.3 μM.

Original language	English (US)
Pages (from-to)	76-78
Number of pages	3
Journal	Moscow University Biological Sciences Bulletin
Volume	66
Issue number	2
DOIs	https://doi.org/10.3103/S0096392511020106
State	Published - Jun 2011
Externally published	Yes

Keywords

Bacillus psychrodurans
DNA methyltrasferase
enzyme kinetics
gene cloning

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@article{505730afaeff49828768ceaa4d260db3,

title = "Cloning and analysis of biochemical and catalytic properties of DNA methyltransferase M1.BspACI",

abstract = "DNA methyltransferases genes of the BspACI restriction-modification system from Bacillus psychrodurans AC have been cloned in E. coli cells. Analysis of amino acid sequences of the proteins showed that both of these genes belong to C5 DNA methyltransferases. Gene M1. BspACI has been subcloned in pJW2 vector. A high-purity recombinant enzyme has been obtained using chromatography on different carriers. It has been shown that M1. BspACI modifies the first cytosine residue in the sequence 5′-CCGC-3′. Kinetic parameters of DNA methylation by the enzyme have been determined. Catalytic constant appears to be 0.095 ± 0.002 min-1. Km phage is λ DNA-0.053 ± 0.007 μM, and KmSAM is 5.1 ± 0.3 μM.",

keywords = "Bacillus psychrodurans, DNA methyltrasferase, enzyme kinetics, gene cloning",

author = "Tarasova, {M. V.} and Kuznetsov, {V. V.} and Netesova, {N. A.} and Gonchar, {D. A.} and Degtyarev, {S. Kh}",

year = "2011",

month = jun,

doi = "10.3103/S0096392511020106",

language = "English (US)",

volume = "66",

pages = "76--78",

journal = "Moscow University Biological Sciences Bulletin",

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publisher = "Allerton Press Inc.",

number = "2",

}

TY - JOUR

T1 - Cloning and analysis of biochemical and catalytic properties of DNA methyltransferase M1.BspACI

AU - Tarasova, M. V.

AU - Kuznetsov, V. V.

AU - Netesova, N. A.

AU - Gonchar, D. A.

AU - Degtyarev, S. Kh

PY - 2011/6

Y1 - 2011/6

N2 - DNA methyltransferases genes of the BspACI restriction-modification system from Bacillus psychrodurans AC have been cloned in E. coli cells. Analysis of amino acid sequences of the proteins showed that both of these genes belong to C5 DNA methyltransferases. Gene M1. BspACI has been subcloned in pJW2 vector. A high-purity recombinant enzyme has been obtained using chromatography on different carriers. It has been shown that M1. BspACI modifies the first cytosine residue in the sequence 5′-CCGC-3′. Kinetic parameters of DNA methylation by the enzyme have been determined. Catalytic constant appears to be 0.095 ± 0.002 min-1. Km phage is λ DNA-0.053 ± 0.007 μM, and KmSAM is 5.1 ± 0.3 μM.

AB - DNA methyltransferases genes of the BspACI restriction-modification system from Bacillus psychrodurans AC have been cloned in E. coli cells. Analysis of amino acid sequences of the proteins showed that both of these genes belong to C5 DNA methyltransferases. Gene M1. BspACI has been subcloned in pJW2 vector. A high-purity recombinant enzyme has been obtained using chromatography on different carriers. It has been shown that M1. BspACI modifies the first cytosine residue in the sequence 5′-CCGC-3′. Kinetic parameters of DNA methylation by the enzyme have been determined. Catalytic constant appears to be 0.095 ± 0.002 min-1. Km phage is λ DNA-0.053 ± 0.007 μM, and KmSAM is 5.1 ± 0.3 μM.

KW - Bacillus psychrodurans

KW - DNA methyltrasferase

KW - enzyme kinetics

KW - gene cloning

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DO - 10.3103/S0096392511020106

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SN - 0096-3925

VL - 66

SP - 76

EP - 78

JO - Moscow University Biological Sciences Bulletin

JF - Moscow University Biological Sciences Bulletin

IS - 2

ER -

Cloning and analysis of biochemical and catalytic properties of DNA methyltransferase M1.BspACI

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