Published online 1 October 2008 Nucleic Acids Research, 2008, Vol. 36, No. 19 6109–6117 doi:10.1093/nar/gkn622 Central base pair flipping and discrimination by PspGI Roman H. Szczepanowski1,2, Michael A. Carpenter3, Honorata Czapinska1,2, Mindaugas Zaremba 4 , Gintautas Tamulaitis4, Virginijus Siksnys4, Ashok S. Bhagwat3 and Matthias Bochtler1,2,5,* 1International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland, 2Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01309 Dresden, Germany, 3Department of Chemistry, Wayne State University, Detroit, MI 48202, USA, 4Institute of Biotechnology, Graiciuno 8, LT-02241, Vilnius, Lithuania and 5Schools of Chemistry and Biosciences, Main Building, Park Place, Cardiff University, Cardiff CF10 3AT, UK Received July 28, 2008; Revised and Accepted September 11, 2008 ABSTRACT PspGI is a representative of a group of restric- tion endonucleases that recognize a pentameric sequence related to CCNGG. Unlike the previously investigated Ecl18kI, which does not have any spe- cificity for the central base pair, PspGI prefers A/T over G/C in its target site. Here, we present a struc- ture of PspGI with target DNA at 1.7 A˚ resolution. In this structure, the bases at the center of the recog- nition sequence are extruded from the DNA and flipped into pockets of PspGI. The flipped thymine is in the usual anti conformation, but the flipped adenine takes the normally unfavorable syn confor- mation. The results of this and the accompanying manuscript attribute the preference for A/T pairs over G/C pairs in the flipping position to the intrin- sically lower penalty for flipping A/T pairs and to selection of the PspGI pockets against guanine and cytosine. Our data show that flipping can con- tribute to the discrimination between normal bases. This adds a new role to base flipping in addition to its well-known function in base modification and DNA damage repair. INTRODUCTION Many DNA binding proteins and enzymes recognize sequences in DNA that are 2-fold symmetric (palin- dromic). DNA duplexes that consist of an odd num- ber of base pairs can be at best approximately symmetric, because exact 2-fold symmetry would require ‘like-with-like’ rather than Watson–Crick base pairing for the central nucleotide pair. Throughout this article, we refer to DNA sequences with an odd number of base pairs, which are symmetric except at the center of the duplex, as ‘pseudopalindromes’. Enzymes that modify both strands of such duplexes must be insensitive to the violation of 2-fold symmetry at the centers of their target sequences. In other words, they cannot distinguish A/T and T/A pairs, but may distinguish them from G/C and C/G pairs. In the usual shorthand notation for DNA bases (W stands for A or T; S stands for G or C; N stands for A, T, G or C), their speciﬁcity for this position must therefore be W, S or N (Figure 1). In order to better understand this form of sequence recognition, we have focused on restriction endonuclease PspGI. This enzyme recognizes the pseudopalindromic sequence CCWGG and cleaves the two strands so that ﬁve nucleotide 50-overhangs result (1). Sequence compar- isons indicate that PspGI is similar to the catalytic domain of EcoRII (2), which has the same speciﬁcity (3,4) and to Ecl18kI, which recognizes the related sequence CCNGG, but displays no speciﬁcity for the central base pair (5). Detailed sequence comparisons and biochemical studies indicate that Ecl18kI, PspGI and the catalytic domain of EcoRII have a shared dimeric architecture and conserved active sites (6–9). So far, only a structure of EcoRII with- out DNA (10) and the crystal structure of Ecl18kI with DNA have been published (11). The Ecl18kI structure indicates that this enzyme ﬂips the central bases of its target sequence into hydrophobic pockets. Signiﬁcant sequence similarity between Ecl18kI and PspGI/EcoRII suggests that these enzymes should also ﬂip the nucleotide at the center of the CCXGG sequence, when X is adenine or thymine, and might or might not ﬂip it when X is *To whom correspondence should be addressed. Tel: +48 22 5970732 (Warsaw); Tel: +44 29 208 70625 (Cardiff); Fax: +48 22 597 0715; Email: mbochtler@iimcb.gov.pl or bochtlerm@cardiff.ac.uk The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. 2008 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.