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# Mutagenesis of Conserved Lysine Residues in Bacteriophage T5 5′-3′ Exonuclease Suggests Separate Mechanisms of Endo- and Exonucleolytic Cleavage

Scott J. Garforth, Thomas A. Ceska, Dietrich Suck and Jon R. Sayers
Proceedings of the National Academy of Sciences of the United States of America
Vol. 96, No. 1 (Jan. 5, 1999), pp. 38-43
Stable URL: http://www.jstor.org/stable/47118
Page Count: 6
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## Abstract

Efficient cellular DNA replication requires the activity of a 5′-3′ exonuclease. These enzymes are able to hydrolyze $\text{DNA}· \text{DNA}$ and $\text{RNA}· \text{DNA}$ 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 angstrom. 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.

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