Restriction endonucleases (REases) occur in all free-living bacteria and archaea and are believed to function to defend their hosts against invasion by foreign DNA, particularly from bacteriophage (Pingoud et al., 2005
). REases vary in sequence, structure, oligomeric composition, substrate-specificity, and enzymatic behavior (Bujnicki, 2003
). They range from compact monomers that act independently, to elaborate multifunctional protein assemblages, and typically recognize target sequences in duplex DNA ranging from four to eight specific base pairs in length. (Pingoud et al., 2005
). These sequences can be symmetric or asymmetric, as well as continuous or discontinuous, depending upon the enzyme architecture.
Several distinct catalytic site motifs and mechanisms have been identified among restriction endonucleases, suggesting this enzymatic and biological function has evolved independently several times. The most common catalytic motif, that of the 'PD…(D/E)xK' nuclease superfamily, is the the most wide-spread and best understood (Kosinski et al., 2005
). Alternative catalytic motifs, associated with quite different core protein folds, have been identified in many additional restriction endonucleases, including the ‘HNH’ (Cymerman et al., 2006
; Jakubauskas et al., 2007
; Saravanan et al., 2004
) and the ‘GIY-YIG’ (Ibryashkina et al., 2007
) motifs (both of which are more commonly associated with mobile homing endonucleases from bacteriophage) (Stoddard, 2005
). All three of these catalytic lineages are also found in a much wider variety of enzymes involved in DNA metabolism and modification, including those responsible for DNA repair, recombination and fidelity (Cymerman et al., 2006
; Dunin-Horkawicz et al., 2006
; Kosinski et al., 2005
). As well, isolated examples of two additional structural motifs (containing the phospholipase D and 'half-pipe' folds) have also observed for R.BfiI and R.PabI, respectively (Grazulis et al., 2005
; Miyazono et al., 2007
The conserved structural core surrounding the HNH motif is termed the 'ββα-metal' fold. This protein topology consists of two anti-parallel β-strands connected by a loop of variable length, flanked by an α-helix (Mehta et al., 2004
),(Kuhlmann et al., 1999
). A binding site for a single catalytic metal ion—typically magnesium—is embedded within this catalytic fold. In some instances, significant insertions of additional structural elements are observed within this motif (Eastberg et al., 2007
; Stoddard, 2005
). The ββα-metal fold can exist as an independently folded catalytic domain (as observed in colicins) or it can be fused to additional protein domains that dictate DNA binding specificity and cleavage activity.
The PD…(D/E)×K motif can be very well-suited for recognition of short DNA sequences with high fidelity, because the catalytic center is surrounded by a densely packed array of side chains that can contact neighboring base pairs in the major groove in a sequence-specific manner (Orlowski and Bujnicki, 2008
). In contrast, the HNH motif and its associated ββα-metal fold appears less well-suited for this task. In order to target the scissile phosphate, the catalytic core motifs of these enzymes primarily interact with the DNA backbone where they contribute little to sequence-specificity and fidelity (Eastberg et al., 2007
). Sequence-recognition by these enzymes is therefore usually carried out by additional protein domains that are tethered to the ββα-metal region, necessitating significant repackaging and augmentation of this catalytic motif.
Recently, the structure of the ββα-metal restriction endonuclease Hpy99I was determined in complex with its DNA substrate, 5' - CGWCG - 3', at 1.5 Å resolution (Sokolowska et al., 2009
) (W=A or T). Hpy99I binds as a homodimer and forms a ring-like structure that encircles the DNA. The protein contacts all four C:G base pairs within both the minor and major groove, and contacts the central base pair (A:T or T:A) in only the minor groove. The DNA is slightly bent in the complex. All nucleotides in the target site are found in canonical Watson-Crick basepair interactions.
In contrast, PacI is a 'rare-cutting' homodimeric HNH restriction endonuclease found in the bacterium Pseudomonas alcaligenes
. It recognizes the symmetric eight base pair duplex DNA sequence 5' – TTAAT/TAA - 3' and cleaves each strand between the internal thymine residues (as the position indicated by "/") to generate product fragments containing 2-base, 3’-overhangs (Roberts et al., 2010
). PacI is one of the smallest REases known, comprising only 142 amino acids per subunit, eight of which are cysteines (). Its gene resides within a super-integron, a chromosomal array that contains multiple gene cassettes each flanked by a large direct repeat sequence and mobilized by a common site-specific integrase (Vaisvila et al., 2001
). Unlike the vast majority of REases, which are accompanied by DNA-methyltransferases that protect the cell’s own DNA from REase auto-digestion, PacI appears to be a solitary enzyme with no companion methyltransferase (see Supplementary Material
). Host protection in this rare instance seems likely to depend not on the methylation of recognition sequences, but rather on the absence of such sequences in the P.alcaligene
The structure of the PacI restriction endonuclease
The length of the PacI recognition sequence (eight basepairs) places this enzyme in the company of NotI and SfiI, two other 'rare-cutting' endonucleases the co-crystal structures of which have been solved (Qiang and Schildkraut, 1987
). NotI and SfiI belong to the PD…(D/E)×K catalytic site superfamily, and in contrast to PacI, recognize sequences composed entirely of G:C base pairs.
Bioinformatics analysis of PacI (Orlowski and Bujnicki, 2008
), and independent analyses with online protein fold prediction servers such as PHYRE (Bennett-Lovsey et al., 2008
) suggest the presence of an HNH-related catalytic site. The likely presence of this motif, combined with the opportunity to compare a ‘rare A:T-cutter’ to two ‘rare G:C-cutters’, led us to determine the structure of PacI bound to DNA. PacI displays little resemblance to either NotI or SfiI, and while it contains structural elements similar to those in Hpy99I, the arrangements of these elements and the overall folds of the two proteins are strikingly different. PacI binding induces an unusual distortion of its DNA target sequence that completely disrupts and reorganizes it normal Watson-Crick duplex structure.