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Nucleic Acids Research Advance Access originally published online on September 26, 2007
Nucleic Acids Research 2007 35(19):6539-6546; doi:10.1093/nar/gkm702
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Nucleic Acids Research, 2007, Vol. 35, No. 19 6539-6546
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

The restriction endonuclease R.NmeDI from Neisseria meningitidis that recognizes a palindromic sequence and cuts the DNA on both sides of the recognition sequence

Agnieszka Kwiatek* and Andrzej Piekarowicz

Institute of Microbiology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland

*To whom the correspondence should be addressed. Tel: +48 22 5541521; Fax: +48 22 5541402; Email: akwiat{at}biol.uw.edu.pl

Received May 28, 2007. Revised August 23, 2007. Accepted August 23, 2007.

The restriction endonuclease Type II R.NmeDI from Neisseria meningitidis 2120 (serogroup C, ST-11 complex) was characterized. The cloned nmeDIR gene was expressed in Escherichia coli cells, and the endonucleolytic and restriction activities of R.NmeDI were then observed in vitro and in vivo. The nmeDIR gene consists of 1056 bp coding 351 aa protein with a calculated molecular weight of M(r) = 39 000 ± 1000 Da. The R.NmeDI enzyme was purified to apparent homogeneity following overexpression, using metal affinity chromatography. This enzyme recognizes a palindrome sequence and cleaves double-stranded DNA upstream and downstream of its recognition sequence (12/7) RCCGGY (7/12) (R = A/G, Y = C/T) cutting out a 25-bp fragment. R.NmeDI cleaves in two steps. The enzyme cleaves the first strand randomly on either side of the recognition sequence generating an intermediate, and the second cleavage occurs more slowly and results in the production of a final reaction product. The R.NmeDI endonuclease requires two recognition sequences for effective cleavage. The tetramer is an active form of the R.NmeDI enzyme.


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