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J. Biol. Chem., Vol. 276, Issue 22, 19172-19181, June 1, 2001

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Biochemical Analysis of Point Mutations in the 5'-3' Exonuclease of DNA Polymerase I of Streptococcus pneumoniae
FUNCTIONAL AND STRUCTURAL IMPLICATIONS^*,

Mónica Amblar, Mario García de Lacoba, Maria A. Corrales, and Paloma López

Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, 28006 Madrid, Spain

To define the active site of the 5'-3' exonucleolytic domain of the Streptococcus pneumoniae DNA polymerase I (Spn pol I), we have constructed His-tagged Spn pol I fusion protein and introduced mutations at residues Asp¹⁰, Glu⁸⁸, and Glu¹¹⁴, which are conserved among all prokaryotic and eukaryotic 5' nucleases. The mutations, but not the fusion to the C-terminal end of the wild-type, reduced the exonuclease activity. The residual exonuclease activity of the mutant proteins has been kinetically studied, together with potential alterations in metal binding at the active site. Comparison of the catalytic rate and dissociation constant of the D10G, E114G, and E88K mutants and the control fusion protein support: (i) a critical function of Asp¹⁰ in the catalytic event, (ii) a role of Glu¹¹⁴ in the exonucleolytic reaction, being secondarily involved in both catalysis and DNA binding, and (iii) a nonessential function of Glu⁸⁸ for the exonuclease activity of Spn pol I. Moreover, the pattern of metal activation of the mutant proteins indicates that none of the three residues is a metal-ligand at the active site. These findings and those previously obtained with D190A mutant of Spn pol I are discussed in relation to structural and mutational data for related 5' nucleases.

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