Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate

AUTOR(ES)

Zharkov, Dmitry O.

FONTE

Oxford University Press

RESUMO

Endonuclease VIII (Nei) of Escherichia coli is a DNA repair enzyme that excises oxidized pyrimidines from DNA. Nei shares with formamidopyrimidine-DNA glycosylase (Fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential β-elimination steps. However, Nei differs significantly from Fpg in substrate specificity. We determined the structure of Nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 Å resolution. The crosslink is derived from a Schiff base intermediate that precedes β-elimination and is stabilized by reduction with NaBH4. Nei consists of two domains connected by a hinge region, creating a DNA binding cleft between domains. DNA in the complex is sharply kinked, the deoxyribitol moiety is bound covalently to Pro1 and everted from the duplex into the active site. Amino acids involved in substrate binding and catalysis are identified. Molecular modeling and analysis of amino acid conservation suggest a site for recognition of the damaged base. Based on structural features of the complex and site-directed mutagenesis studies, we propose a catalytic mechanism for Nei.

Documentos Relacionados

• Structure of a trapped endonuclease III–DNA covalent intermediate
• Characterization of endonuclease III (nth) and endonuclease VIII (nei) mutants of Escherichia coli K-12.
• Escherichia coli endonuclease VIII: cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants.
• A Novel Role for Escherichia coli Endonuclease VIII in Prevention of Spontaneous G→T Transversions
• The genes encoding endonuclease VIII and endonuclease III in Escherichia coli are transcribed as the terminal genes in operons