Mutagenesis of conserved lysine residues in bacteriophage T5 5′-3′ exonuclease suggests separate mechanisms of endoand exonucleolytic cleavage
| Autor: | Dietrich Suck, Thomas A. Ceska, Scott J. Garforth, Jon R. Sayers |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1999 |
| Předmět: |
Exonuclease
Models Molecular Exodeoxyribonuclease V Biology Crystallography X-Ray Conserved sequence chemistry.chemical_compound Endonuclease Amino Acid Sequence Endodeoxyribonucleases Bacteriophage T5 Conserved Sequence RecBCD Multidisciplinary Hydrolysis Lysine Active site DNA Biological Sciences Hydrogen-Ion Concentration biology.organism_classification DNA-Binding Proteins Exodeoxyribonucleases Biochemistry chemistry biology.protein Mutagenesis Site-Directed T-Phages |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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