Complementation between inactive fragments of SssI DNA methyltransferase

BMC Molecular Biology

Antal Kiss¹  Edit Tímár¹  Krystyna Ślaska-Kiss¹ 

[1] Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Temesvári krt. 62, 6726, Szeged, Hungary

关键词: Zinc finger;    Protein fusion;    Protein fragment complementation;    5-methylcytosine;    DNA methylation;    SssI DNA methyltransferase;   

Others  :  1091946
DOI  :  10.1186/1471-2199-13-17

 received in 2012-03-02, accepted in 2012-05-30,  发布年份 2012

【 摘 要 】

Background

Silencing mammalian genes by targeted DNA (cytosine-5) methylation of selected CG sites in the genome would be a powerful technique to analyze epigenomic information and to study the roles of DNA methylation in physiological and pathological states. A promising approach of targeted DNA methylation is based on the ability of split fragments of a monomeric DNA methyltransferase (C5-MTase) to associate and form active enzyme. A few C5-MTases of different specificities have been shown to possess the ability of fragment complementation, but a demonstration of this phenomenon for a C5-MTase, which has CG specificity and thus can be targeted to methylate any CG site, has been lacking. The purpose of this study was to test whether the CG-specific prokaryotic C5-MTase M.SssI shows the phenomenon of fragment complementation.

Results

We show that truncated inactive N-terminal fragments of M.SssI can assemble with truncated inactive C-terminal fragments to form active enzyme in vivo when produced in the same E. coli cell. Overlapping and non-overlapping fragments as well as fragments containing short appended foreign sequences had complementation capacity. In optimal combinations C-terminal fragments started between conserved motif VIII and the predicted target recognizing domain of M.SssI. DNA methyltransferase activity in crude extracts of cells with the best complementing fragment pairs was ~ 4 per cent of the activity of cells producing the full length enzyme. Fusions of two N-terminal and two C-terminal fragments to 21.6 kDa zinc finger domains only slightly reduced complementation ability of the fragments.

Conclusions

The CG-specific DNA methyltransferase M.SssI shows the phenomenon of fragment complementation in vivo in E. coli. Fusion of the split fragments to six unit zinc finger domains does not substantially interfere with the formation of active enzyme. These observations and the large number of complementing fragment combinations representing a wide range of MTase activity offer the possibility to develop M.SssI into a programmable DNA methyltransferase of high specificity.

【 授权许可】

   
2012 Slaska-Kiss et al.; licensee BioMed Central Ltd.

【 图 表 】

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