Albany 2001

category image Biomolecular
SUNY at Albany
June 19-23, 2001

The structure and active site of the DNA junction-resolving enzyme T7 endonuclease I

Endonuclease I is a junction-resolving enzyme encoded by bacteriophage T7, that selectively binds and cleaves four-way DNA junctions. We have solved the structure of this dimeric enzyme at atomic resolution by X-ray crystallography at 2.1 A resolution. The protein forms a strong symmetrical dimer arranged in two separated domains. Each domain is composed of elements from both monomers. The active site comprises the side chains of three acidic amino acids (Glu 20, Asp 55 and Glu 65) together with a lysine (Lys 67), and shares strong similarities with a number of type II restriction enzymes. However, it differs from a typical restriction enzyme as the proposed catalytic residues in both active sites are contributed by both polypeptides of the dimer. Mutagenesis experiments confirm the importance of all the proposed active site residues, and in vitro complementation experiments using heterodimers formed from mutants in different active site residues demonstrate that Glu 20 is located on a different monomer from the remaining amino acids comprising the active site. The composite active site structure has been confirmed by the use of a cruciform substrate. We propose a two-metal ion mechanism for the hydrolytic cleavage of DNA junctions.

References and Footnotes
  1. J.M. Hadden, M.A. Convery, A.-C. Declais, D.M.J. Lilley and S.E.V. Phillips Crystal structure of the Holliday junction-resolving enzyme T7 endonuclease I at 2.1 A resolution. Nature Struct. Biol. 8, 62-67 (2001).
  2. A.-C. Declais, J. Hadden, S.E.V. Phillips and D.M.J. Lilley The active site of the junction-resolving enzyme T7 endonuclease I. J. Molec. Biol. In the press.

Anne-Cecile Declais (1), Jon Hadden (2), Simon Phillips (2) and David M.J. Lilley (1)

CRC Nucleic Acid Structure Research Group (1), Biochemistry Department, University of Dundee, Dundee DD1 5EH, UK; School of Biochemistry and Molecular Biology (2), University of Leeds, Leeds LS2 9JT. UK.
Email: dmjlilley@bad.dundee.ac.uk, Tel 44-1382-344243