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Nucleic Acids Research 2005 33(15):4775-4787; doi:10.1093/nar/gki787
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Published online 24 August 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

Alice Sears, Luke J. Peakman, Geoffrey G. Wilson1 and Mark D. Szczelkun*

DNA-Protein Interactions Unit, Department of Biochemistry, University of Bristol Bristol, BS8 1TD, UK 1New England Biolabs Inc. 32 Tozer Road, Beverly, MA 01915, USA

*To whom correspondence should be addressed. Tel: +44 0 117 928 7439; Fax: +44 0 117 928 8274; Email: mark.szczelkun{at}bristol.ac.uk

Received June 16, 2005. Revised July 18, 2005. Accepted August 8, 2005.

A new Type III restriction endonuclease designated PstII has been purified from Providencia stuartii. PstII recognizes the hexanucleotide sequence 5'-CTGATG(N)25-26/27-28-3'. Endonuclease activity requires a substrate with two copies of the recognition site in head-to-head repeat and is dependent on a low level of ATP hydrolysis (~40 ATP/site/min). Cleavage occurs at just one of the two sites and results in a staggered cut 25–26 nt downstream of the top strand sequence to generate a two base 5'-protruding end. Methylation of the site occurs on one strand only at the first adenine of 5'-CATCAG-3'. Therefore, PstII has characteristic Type III restriction enzyme activity as exemplified by EcoPI or EcoP15I. Moreover, sequence asymmetry of the PstII recognition site in the T7 genome acts as an historical imprint of Type III restriction activity in vivo. In contrast to other Type I and III enzymes, PstII has a more relaxed nucleotide specificity and can cut DNA with GTP and CTP (but not UTP). We also demonstrate that PstII and EcoP15I cannot interact and cleave a DNA substrate suggesting that Type III enzymes must make specific protein–protein contacts to activate endonuclease activity.

Present address: Luke J. Peakman, Dairy Crest Limited, Station Yard, Totnes, Devon TQ9 5JP, UK


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