Separating substrate recognition from base hydrolysis in human thymine DNA glycosylase by mutational analysis

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Human thymine DNA glycosylase (TDG) initiates base excision repair by removing thymine or uracil from G:T and G:U mismatches.
• Structural analysis of an E. coli homologue identified critical residues for substrate interaction and base hydrolysis.

Purpose of the Study:

• To investigate structure-function relationships in human TDG through mutational analysis.
• To validate the applicability of the E. coli TDG structure-function model to human TDG.

Main Methods:

• Site-directed mutagenesis of human TDG, including N140A and Met-269 mutations.
• Enzyme activity assays to assess substrate binding and base removal.
• Comparative analysis with E. coli TDG structure-function models.

Main Results:

• The N140A mutation abolished base removal while retaining substrate binding.
• Mutation of Met-269 impaired stable substrate binding but not glycosylase activity.
• Evidence suggests G:U mismatches are preferred substrates for both recognition and hydrolysis.
• Stable complementary strand interactions are crucial for TDG's hydrolytic efficiency.

Conclusions:

• The structure-function model for E. coli TDG is largely applicable to human TDG.
• Specific residues are critical for distinct TDG functions: N140 for catalysis and Met-269 for stable binding.
• Human TDG shows a preference for G:U mismatches and relies on strand interactions to enhance its hydrolytic potential.