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.