DNA binding and cleavage by the periplasmic nuclease Vvn: A novel structure with a known active site

Chia-Lung Li^1,2, Lien-I Hor³, Zee-Fen Chang¹, Li-Chu Tsai², Wei-Zen Yang² and

Hanna S. Yuan^1,2

¹Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC.

²Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, ROC.

³Department of Microbiology and Immunology, College of Medicine, National Cheng-Kung University, Tainan, Taiwan, ROC.

Abstract

The Vibrio vulnificus nuclease, Vvn, is a non-specific periplasmic nuclease capable of digesting DNA and RNA.  The crystal structure of Vvn and that of Vvn mutant H80A in complex with DNA were resolved at 2.3 Å resolution.  Vvn has a novel mixed a/b topology containing four disulfide bridges, suggesting that Vvn is not active under reducing conditions in the cytoplasm.  The overall structure of Vvn shows no similarity to other endonucleases, however, a known “bba-metal” motif is identified in the central cleft region.  The crystal structure of the mutant Vvn/DNA complex demonstrates that Vvn binds mainly at the minor groove of DNA, resulting in duplex bending towards the major groove by about 20^o.  Only the DNA phosphate backbones make hydrogen bonds with Vvn, suggesting structural basis for its sequence-independent recognition of DNA and RNA.  Based on the enzyme/substrate and enzyme/product structures observed in the mutant Vvn/DNA crystals, a catalytic mechanism is proposed.  This structural study suggests that Vvn hydrolyzes DNA by a general single-metal ion mechanism, and indicates how non-specific DNA-binding proteins may recognize DNA.

EMBO J. 22, 4014-4025 (2003)