Engineering functional changes in Escherichia coli endonuclease III based on phylogenetic and structural analyses.

Area of Science:

• Biochemistry
• Molecular Biology
• DNA Repair Mechanisms

Background:

• Escherichia coli endonuclease III (EcoNth) is crucial for removing oxidative pyrimidine DNA damage.
• EcoNth possesses dual DNA glycosylase and apurinic/apyrimidinic (AP) lyase activities.
• Specific amino acids (Ser39, Asp44, Arg184) are conserved in EcoNth and hypothesized to be vital for catalysis.

Purpose of the Study:

• To investigate the roles of EcoNth's Ser39, Asp44, and Arg184 in DNA glycosylase and AP lyase activities.
• To understand how these residues contribute to base recognition and catalytic function.
• To elucidate the functional consequences of specific amino acid substitutions by creating mutants.

Main Methods:

• Phylogenetic analysis of natural sequences to identify conserved amino acids.
• Site-directed mutagenesis to create EcoNth mutants: S39L, D44V, and R184A.
• Biochemical assays to measure glycosylase and AP lyase activities of wild-type and mutant EcoNth.

Main Results:

• EcoNth S39L mutant showed reduced glycosylase activity but retained AP lyase activity.
• EcoNth D44V mutant retained glycosylase activity but had significantly impaired AP lyase activity, indicating Asp44's role in beta-elimination.
• EcoNth R184A mutant maintained lyase activity but displayed altered glycosylase specificity.

Conclusions:

• Phylogeny-guided mutagenesis identified critical roles for Ser39, Asp44, and Arg184 in EcoNth's substrate binding and catalytic efficiency.
• Asp44 is essential for the beta-elimination step of the AP lyase activity.
• These findings provide insights into base recognition and catalysis within the EcoNth active site, aided by structural context.