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Nucleic Acids Research 2004 32(21):6187-6199; doi:10.1093/nar/gkh958
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Published online 29 November 2004

Nucleic Acids Research, Vol. 32 No. 21 © Oxford University Press 2004; all rights reserved

Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification

Siu-hong Chan, Zhenyu Zhu, James L. Van Etten1 and Shuang-yong Xu*

New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA and 1 Department of Plant Pathology and Nebraska Center for Virology, University of Nebraska, Lincoln, NE 68583, USA

* To whom correspondence should be addressed. Tel: +1 978 927 7287; Fax: +1 978 921 1350; Email: xus{at}neb.com

Received October 15, 2004; Revised and Accepted November 5, 2004

The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG^CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65°C with a temperature optimum of 30–45°C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.


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S.-Y. Xu, Z. Zhu, P. Zhang, S.-H. Chan, J. C. Samuelson, J. Xiao, D. Ingalls, and G. G. Wilson
Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI
Nucleic Acids Res., July 9, 2007; 35(14): 4608 - 4618.
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