Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo- and exonucleolytic cleavage

S. J. Garforth, T. A. Ceska, D. Suck, J. R. Sayers

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26 Scopus citations

Abstract

Efficient cellular DNA replication requires the activity of a 5'-3' exonuclease. These enzymes are able to hydrolyze DNA·DNA and RNA·DNA substrates exonucleolytically, and they are structure-specific endonucleases. The 5'-3' exonucleases are conserved in organisms as diverse as bacteriophage and mammals. Crystal structures of three representative enzymes identify two divalent-metal-binding sites typically separated by 8-10 Å. Site-directed mutagenesis was used to investigate the roles of three lysine residues (K83, K196, and K215) situated near two metal-binding sites in bacteriophage T5 5'- 3' exonuclease. Neither K196 nor K215 was essential for either the exo- or the endonuclease activity, but mutation of these residues increased the dissociation constant for the substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A). Biochemical analysis demonstrated that K83 is absolutely required for exonucleolytic activity on single-stranded DNA but is not required for endonucleolytic cleavage of flap structures. Structural analysis of this mutant by x-ray crystallography showed no significant perturbations around the metal-binding sites in the active site. The wild-type protein has different pH optima for endonuclease and exonuclease activities. Taken together, these results suggest that different mechanisms for endo- and exonucleolytic hydrolysis are used by this multifunctional enzyme.

Original languageEnglish (US)
Pages (from-to)38-43
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number1
DOIs
StatePublished - Jan 5 1999
Externally publishedYes

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