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  The Cfr10I restriction enzyme is functional as a tetramer

Siksnys, V., Skirgaila, R., Sasnaukas, G., Urbanke, C., Cherny, D. I., Grazulis, S., et al. (1999). The Cfr10I restriction enzyme is functional as a tetramer. Journal of Molecular Biology, 291, 1105-1118.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0012-FBBD-6 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0027-E61A-F
Genre: Journal Article

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 Creators:
Siksnys, V., Author
Skirgaila, R., Author
Sasnaukas, G., Author
Urbanke, C., Author
Cherny, D. I.1, Author              
Grazulis, S.2, Author
Huber, R.2, Author
Affiliations:
1Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society, ou_578628              
2Max Planck Society, ou_persistent13              

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Free keywords: Cfr10I; quaternary structure; restriction enzyme; site-directed mutagenesis
 Abstract: It is thought that most of TypeII restriction endonucleases interact with DNA as homodimers. The Cfr10I is a typical TypeII restriction enzyme that recognises 5’-PuØCCGGPy sequence and cleaves it as indicated by the arrow. Gel-filtration and analytical ultracentrifugation data presented here indicate that Cfr10I is a homotetramer in solution. The only SfiI restriction enzyme that recognises long interrupted recognition sequence 5’-GGCCNNNNNGGCC has been previously reported to operate as a tetramer, however its structure is unknown. Analysis of Cfr10I crystals revealed that a single molecule in the asymmetric unit is repeated by D2 symmetry to form a tetramer. To determine whether the packing of the Cfr10I in the crystal reflects the quaternary structure of the protein in solution, the tryptophan W220 residue located at the putative dimer-dimer interface was mutated to alanine and the structural and functional consequences of the substitution were analysed. Equilibrium sedimentation experiments revealed that in contrast to the wild type Cfr10I, the W220A mutant exists in solution predominantly as a dimer. In addition, the tetramer seems to be a catalytically important form of Cfr10I since the DNA cleavage activity of the W220A mutant is <0.1% of that of the wild type enzyme. Further, analysis of plasmid DNA cleavage suggests that the Cfr10I tetramer is able to interact with two copies of the recognition sequence, located on the same DNA molecule. Indeed, electron microscopy studies demonstrated that two distant recognition sites are brought together through the DNA looping induced by the simultaneous binding of the Cfr10I tetramer to both sites. These data are consistent with the tetramer being a functionally important form of Cfr10I.

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Language(s): eng - English
 Dates: 2005-08-051999
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Method: -
 Identifiers: eDoc: 236906
Other: 11480
 Degree: -

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Title: Journal of Molecular Biology
Source Genre: Journal
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Publ. Info: -
Pages: - Volume / Issue: 291 Sequence Number: - Start / End Page: 1105 - 1118 Identifier: -