The 3′-5′ exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.

Abstract

We have used site‐directed mutagenesis to change amino acid side chains that have been shown crystallographically to be in close proximity to a DNA 3′ terminus bound at the 3′‐5′ exonuclease active site of Klenow fragment. Exonuclease assays of the resulting mutant proteins indicate that the largest effects on exonuclease activity result from mutations in a group of carboxylate side chains (Asp355, Asp424 and Asp501) anchoring two divalent metal ions that are essential for exonuclease activity. Another carboxylate (Glu357) within this cluster seems to be less important as a metal ligand, but may play a separate role in catalysis of the exonuclease reaction. A second group of residues (Leu361, Phe473 and Tyr497), located around the terminal base and ribose positions, plays a secondary role, ensuring correct positioning of the substrate in the active site and perhaps also facilitating melting of a duplex DNA substrate by interacting with the frayed 3′ terminus. The pH‐dependence of the 3′‐5′ exonuclease reaction is consistent with a mechanism in which nucleophilic attack on the terminal phosphodiester bond is initiated by a hydroxide ion coordinated to one of the enzyme‐bound metal ions.

Structural aspects of protein-DNA recognition

“…Two divalent metal ions are also seen interacting with the phosphate of the 3' terminal base and are thought to be involved in the positioning and cleavage of the phosphodiester bond (Derbyshire et al, 1988;Freemont et al, 1988). Recent site-directed mutagenesis studies of all the amino acids implicated by the crystal structure to be involved in substrate binding or catalysis are in agreement with the structural observations (Derbyshire et al, 1991). The relationship between the polymerase and 3'-5' exonuclease active sites, which are some 25 A apart, remains unclear.…”

Differential Incorporation and Removal of Antiviral Deoxynucleotides by Human DNA Polymerase γ

“…DNA polymerase ␥ is a family A polymerase, which is best typified by the Escherichia coli pol I (32). Mutagenesis studies and the three-dimensional structure of the E. coli pol I exonuclease active site show that the 3Ј-OH group of the terminal nucleotide plays a key role in the exonucleolytic mechanism by forming a hydrogen bond with a glutamic acid side chain (Glu-200 in human pol ␥) facilitating this residue to makes an ionic bond to the catalytic Mg 2ϩ and a hydroxide anion (33)(34)(35). Stabilized by the metal cation, the activated hydroxide anion attacks the phosphodiester bond, inverting the configuration of the phosphate to release of the terminal nucleoside monophosphate.…”

Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis

“…6 and 7B, together with sequence comparisons of CoV ExoN domains with DEDD family exonucleases (13), and the biochemical and structural information available for several of these cellular homologs (24)(25)(26)(27)(28)(29), suggest that the ExoN activity critically depends on two divalent metal ions that are coordinated by the SARS-CoV pp1ab residues Asp-5992 and Glu-5994 (both from Exo I), Asp-6145 (Exo II), and Asp-6175 (Exo III). With few exceptions (30), the conserved Exo I-III Asp and Glu residues are known to be essential for DEDD nuclease activity, whereas replacements of the conserved Exo III Tyr͞His residue appear to be slightly less detrimental (26,31,32). The Exo II and Exo III Asp residues (6145 and 6175) proved to be essential for the SARS-CoV ExoN activity, whereas the D5992A͞E5994A and H6170A substitutions resulted in substantially diminished (but still detectable) activities (see above).…”