Nucleic Acids Research, 2005, Vol. 33, No. 15 4775–4787 doi:10.1093/nar/gki787 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 and 1New England Biolabs Inc., 32 Tozer Road, Beverly, MA 01915, USA Received June 16, 2005; Revised July 18, 2005; Accepted August 8, 2005 ABSTRACT A new Type III restriction endonuclease designated PstII has been purified from Providencia stuartii. PstII recognizes the hexanucleotide sequence 50-CTGATG(N)25-26/27-28-30. 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 50-protruding end. Methylation of the site occurs on one strand only at the first adenine of 50-CATCAG-30. 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 gen- ome 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 speci- ficity 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. INTRODUCTION Of the three major Restriction-Modiﬁcation (RM) systems, the Type III enzymes are the least well characterized. A combina- tion of genetics and bioinformatics has revealed, to-date (1), >140 conﬁrmed and putative Type III RM systems not includ- ing orphan open reading frames (ORFs). These represent 6% of all RM systems, although, the true percentage may be higher due to the over-representation of commercially exploit- able Type II enzymes. The vast majority of biochemical studies on Type III RM enzymes have been undertaken using two closely related protein complexes, EcoPI (Please note that by common agreement the nomenclature EcoPI replaces the alternatives EcoP1I and EcoP1) and EcoP15I (2–4). Other enzymes that have been analysed to a lesser degree include HinfIII, HineI (5–7), StyLTI (8,9), LlaFI (10), BceS1 (11) and PhaB1 (1,12). A general model for DNA modiﬁcation and cleavage by all Type III enzymes has been proposed (2–4), based partly on a general model that has been established for the related Type I RM enzymes (3). In contrast to these uniﬁed Type I and III schemes, the current view of the Type II RM enzymes is that the DNA cleavage is accomplished by a multiplicity of different mech- anisms, many of which are very different from the activities of archetypal enzymes such as EcoRI and EcoRV (13). Indeed it now appears that the majority of Type II enzymes must bind to multiple recognition sequences in order to cut DNA. This new orthodoxy has only been revealed by the analysis of an expan- ded range of enzyme systems [e.g. even when recognizing the same DNA sequence, many different Type II mechanisms have evolved, (14)]. Therefore, one could question whether it is appropriate to invoke a single mechanistic model for all Type III enzymes given the paucity of in-depth comparative studies. Do alternative Type III activities exist? Here, and in the accompanying paper (15), we describe the detailed char- acterization of a new Type III RM system, the PstII restriction enzyme from Providencia stuartii. From our results it appears that the PstII RM system conforms to the general Type III model and historical evidence of its prototypical Type III restriction activity can be inferred from the genome sequences of some bacteriophage. MATERIALS AND METHODS DNA substrates Unless stated otherwise, all DNA manipulations were carried out using standard procedures (16). All oligonucleotides were *To whom correspondence should be addressed. Tel: +44 0 117 928 7439; Fax: +44 0 117 928 8274; Email: mark.szczelkun@bristol.ac.uk Present address: Luke J. Peakman, Dairy Crest Limited, Station Yard, Totnes, Devon TQ9 5JP, UK The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org