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PvuII endonuclease contains two calcium ions in active sites
1Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.
Journal of Molecular Biology
|July 25, 2000
Summary
Restriction endonucleases like PvuII utilize calcium (Ca2+) for DNA cleavage, revealing a distinct two-metal mechanism. This structural insight clarifies cofactor differences in DNA-cutting enzymes.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biology
Background:
- Restriction endonucleases are enzymes that cut DNA and often require metal cofactors.
- Despite similar catalytic region structures, different endonucleases use distinct metal ions.
Purpose of the Study:
- To investigate the interaction of endonuclease PvuII with calcium (Ca2+), a catalytically incompetent cation.
- To elucidate the mechanism of DNA cleavage by PvuII using Ca2+.
Main Methods:
- Characterization of the PvuII-DNA complex structure in the presence of Ca2+.
- Crystallographic analysis of a glutaraldehyde-crosslinked complex at pH 6.5.
Main Results:
- The structure supports a two-metal ion mechanism for PvuII DNA cleavage.
- The first Ca2+ ion position is conserved among endonucleases.
- The second Ca2+ ion position in PvuII differs from BamHI/BglI and EcoRV, impacting interactions with the DNA scissile phosphate and leaving group.
Conclusions:
- PvuII employs a two-metal mechanism with Ca2+ that shares similarities with BamHI and BglI but differs from EcoRV models.
- The distinct positioning of the second metal ion influences the catalytic mechanism of PvuII.
- Understanding these differences aids in characterizing endonuclease cofactor specificity.