PspGI, a Type II Restriction Endonuclease from the Extreme Thermophile Pyrococcus sp.: Structural and Functional Studies to Investigate an Evolutionary Relationship with Several Mesophilic Restriction Enzymes

Abstract We present here the first detailed biochemical analysis of an archaeal restriction enzyme. Psp GI shows sequence similarity to Sso II, Eco RII, Ngo MIV and Cfr 10I, which recognize related DNA sequences. We demonstrate here that Psp GI, like Sso II and unlike Eco RII or Ngo MIV and Cfr 10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. Psp GI and Sso II differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl 2 and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that Psp GI and Sso II have a very similar DNA binding site and catalytic center as Ngo MIV and Cfr 10I (whose crystal structures are known), and presumably also as Eco RII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with Psp GI, Sso II and Eco RII in the region of the presumptive active site. These results are discussed in an evolutionary context.