6116 Nucleic Acids Research, 2008, Vol. 36, No. 19 than cytosine. On the ‘sugar edge’, thymine and cytosine both have a 2-oxo group and are therefore diﬃcult to distinguish. On the Watson–Crick edge of the ﬂipped thy- mine the O4 atom interacts with a water molecule, and on its ‘C–H’ edge the C5 methyl group is surrounded by a fairly hydrophobic environment. Replacement of thy- mine with cytosine would convert the O4 hydrogen bond acceptor to an N4 hydrogen bond donor, but a hydrogen bond to the water in the same position could still be made. The replacement would also abolish some hydrophobic interactions between the C5 methyl group of thymine and PspGI. Finally, it would convert the N3 atom of the pyrimidine from a hydrogen bond donor to an acceptor and therefore change the favor- able interaction (despite non-ideal geometry) with the car- bonyl oxygen atom of Glu60 into an unfavorable one (Figure 5). Pocket specificity for purines The biochemical data on PspGI suggest that adenine ﬁts the PspGI pocket better than guanine (16). On the ‘sugar edge’, guanine and adenine are distinguishable by the pre- sence or absence of a 2-amino group on the purine ring. In order to test the eﬀect of the 2-amino group, we super- imposed a guanine base on the ﬂipped adenine base in the PspGI-DNA co-crystal structure (Supplementary Figure S2). A few short contacts between the guanine base and amino acid residues in the protein pocket result, but these could be easily relieved by a minor repo- sitioning of the base or by a slight relaxation of the PspGI structure. On the Hoogsteen edge of the ﬂipped purine (which would be located on the major groove side in reg- ular DNA), we found a water molecule bound to the car- bonyl oxygen atom of Glu60 and the 6-amino group and N7 nitrogen atom of the ﬂipped adenine. This interaction could favor adenine over guanine, because a water mole- cule, unless it is protonated, would not allow the same network of hydrogen bonds if the adenine base was replaced with a guanine base containing 6-oxo group (Figure 5). Models for discrimination between W and S PspGI distinguishes A/T pairs from G/C pairs with almost million-fold speciﬁcity; whereas, the related enzyme Ecl18kI does not make this distinction at all (16). We speculated that diﬀerence in the pocket walls might account for this diﬀerence and therefore mutated Phe64 in PspGI to a tryptophan as in Ecl18kI or to alanine as a control, but both mutants retained speciﬁcity for CCWGG sites and did not cleave CCSGG sites (data not shown). Fortunately, some conclusions about PspGI speciﬁcity can be drawn without experimentally convert- ing PspGI into an enzyme with Ecl18kI-like activity and vice versa. The biochemical results suggest that PspGI senses dif- ferences between A/T and G/C pairs already in the bind- ing step and greatly ampliﬁes them in the catalytic step (16). Qualitatively, the disruption of an A/T pair with two Watson–Crick hydrogen bonds is ‘easier’ than the disrup- tion of a G/C pair with three hydrogen bonds, even though stacking interactions contribute to base pair stabi- lity as well (30). Quantitative experiments show that enthalpic eﬀects are partially oﬀset by entropic eﬀects (31), but the general conclusion that A/T base pairs are easier to ﬂip than G/C pairs still holds. In the absence of an enzyme, this is reﬂected by diﬀerences in the rates and equilibrium constants for spontaneous ﬂipping of A/T pairs and G/C pairs (32). We believe that these diﬀerences also explain (at least in part) the diﬀerent binding con- stants for the interaction of PspGI with CCWGG and CCSGG duplexes. The data for the cleavage rates of oligoduplexes with G/C, G/G and C/C instead of the A/T pair at the center of the recognition sequence indicate that the PspGI pocket selects against guanine, and to a lesser extent also against cytosine (16). According to the crystallographic data and 2AP cleavage experiments (12,16), no single feature of cytosine or guanine has ‘veto’ power over PspGI cleavage and can alone explain PspGI speciﬁcity. It rather seems that weak and dispersed interactions between the ﬂipped base and PspGI pocket collectively ensure discrimination against guanine and cytosine. In the absence of a crystal structure of PspGI in com- plex with a non-substrate oligoduplex with G/C instead of the A/T, the drastic diﬀerences in PspGI catalytic rates could have at least three diﬀerent explanations. Guanine and cytosine in the central position could not be ﬂipped at all, ﬂipped only transiently, or stably ﬂipped in a manner that indirectly inhibits DNA cleavage. The ﬁrst possibility, a failure of PspGI to ﬂip guanine or cytosine entirely, appears unlikely, because it would interfere with the com- pression of the DNA and its ﬁt to PspGI, and should therefore decrease the binding constant for the duplex with central G/C more drastically than experimentally observed. Moreover, the fast deamination rate of cytosine in the CCSGG sequence (14) and its accessibility to chlor- oacetaldehyde modiﬁcation (15) suggest that PspGI can unstack this base, even if it does not unstack guanine. For the second possibility, it remains to be explained why transient ﬂipping suppresses catalytic rates far more drastically than the binding constants. The third possibi- lity, binding of the ﬂipped bases to the PspGI pockets in a manner that either allosterically inactivates the enzyme or distorts the phosphodiester backbone remains to be recon- ciled with the distances of the PspGI pockets to the active sites. In other words, the way in which transient or incor- rect stable ﬂipping of bases within the central base pair prevents catalysis at distant scissile phosphates remains unclear. Our explanation for the PspGI speciﬁcity has much in common with interpretation of the speciﬁcity of uracil DNA glycosylase (UDG) and other DNA repair enzymes (33). Like PspGI, these enzymes also ﬂip nucleotides and distinguish lesions with much higher speciﬁcity than would be expected on the basis of base pair strength alone. However, in contrast to these well-studied DNA repair enzymes, PspGI is speciﬁc for natural, undamaged base pairs in regular hydrogen bonding arrangements prior to the ﬂipping event. Consequently, its sequence speciﬁcity is even more remarkable.