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Cloning, overexpression, and biochemical characterization of the catalytic domain of MutY
1Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1071, USA.
Abstract:
Proteolysis of MutY with trypsin indicated that this DNA mismatch repair enzyme could exist as two modules and that the N-terminal domain (Met1-Lys225), designated as p26, could serve as the catalytic domain [Manuel et al. (1996) J. Biol. Chem. 271, 16218-16226]. In this study, the p26 domain has been cloned, overproduced, and purified to homogeneity. Synthetic DNA duplexes containing mismatches, generated with regular bases and nucleotide analogs containing altered functional groups, have been used to characterize the substrate specificity and mismatch repair efficiency of p26. In general, p26 recognized and cleaved most of the substrates which were catalyzed by the intact protein. However, p26 displayed enhanced specificity for DNA containing an inosine. guanine mismatch, and the specificity constant (Kcat/Km) was 2-fold higher. The truncated MutY was able to cleave DNA containing an abasic site with equal efficiency. Dissociation constants (Kd) were obtained for p26 on noncleavable DNA substrates containing a tetrahydrofuran (abasic site analog) or a reduced abasic site. p26 bound these substrates with high specificity, and the Kd values were 3-fold higher when compared to the intact MutY. p26 contains both DNA glycosylase and AP lyase activities, and we provide evidence for a reaction mechanism that proceeds through an imino intermediate. Thus, we have shown for the first time that deletion of 125 amino acids at the C-terminus of MutY generates a stable catalytic domain which retains the functional identity of the intact protein.
Insights
The N-terminal domain of MutY (p26) functions as a stable catalytic domain, retaining DNA repair activity. This truncated enzyme shows enhanced specificity for certain mismatches and binds abasic sites with high affinity.
Area of Science:
- Molecular Biology
- Enzymology
- DNA Repair Mechanisms
Background:
- The DNA mismatch repair enzyme MutY is crucial for maintaining genomic stability.
- Previous studies suggested MutY might consist of distinct functional modules.
Purpose of the Study:
- To investigate the catalytic activity and substrate specificity of the N-terminal domain of MutY (p26).
- To determine if p26 represents a stable and functional catalytic domain.
Main Methods:
- Cloning, overexpression, and purification of the p26 domain.
- Characterization using synthetic DNA duplexes with various mismatches and abasic site analogs.
- Enzyme kinetics and binding assays to determine specificity constants (Kcat/Km) and dissociation constants (Kd).
Main Results:
- The purified p26 domain exhibited DNA glycosylase and AP lyase activities, similar to intact MutY.
- p26 showed enhanced specificity for DNA containing an inosine-guanine mismatch (2-fold higher Kcat/Km).
- p26 bound to abasic site analogs with higher specificity (3-fold higher Kd) compared to intact MutY.
Conclusions:
- The N-terminal domain (p26) of MutY is a stable catalytic domain that retains essential DNA repair functions.
- Deletion of the C-terminal region generates a functional catalytic domain with altered substrate specificity and binding affinity.
- This finding provides insights into the modular structure and catalytic mechanisms of MutY.