PvuII endonuclease contains two calcium ions in active sites

Restriction endonucleases differ in their use of metal cofactors despite ha ving remarkably similar folds for their catalytic regions. To explore this, we have characterized the interaction of endonuclease PvuII with the catal ytically incompetent cation Ca2+. The structure of a glutaralde-hyde-crossl inked crystal of the endonuclease PvuII-DNA complex, determined in the pres ence of Ca2+ at a pH of approximately 6.5, supports a two-metal mechanism o f DNA cleavage by PvuII. The first Ca2+ position matches that found in all structurally examined endonucleases, while the second position is similar t o that of EcoRV but is distinct from that of BamHI and BglI. The location o f the second metal in PvuII, unlike that in BamHI/BglI, permits no direct i nteraction between the second metal and the O3' oxygen leaving group. Howev er, the interactions between the DNA scissile phosphate and the metals, the first metal and the attacking water, and the attacking water and DNA are t he same in PvuII as they are in the two-metal models of BamHI and BglI, but are distinct from the proposed three-metal or the two-metal models of EcoR V. (C) 2000 Academic Press.