Cloning, overexpression, and biochemical characterization of the catalytic domain of MutY

Raymond C. Manuel, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

72 Citations (Scopus)

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 p261. 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 (K(cat)/K(m)) was 2-fold higher. The truncated MutY was able to cleave DNA containing an abasic site with equal efficiency. Dissociation constants (K(d)) were obtained for p26 on nonclearable DNA substrates containing a tetrahydrofuran (abasic site analog) or a reduced abasic site. p26 bound these substrates with high specificity, and the K(d) 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.

Original languageEnglish (US)
Pages (from-to)11140-11152
Number of pages13
JournalBiochemistry
Volume36
Issue number37
DOIs
StatePublished - Sep 16 1997
Externally publishedYes

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Cloning
Organism Cloning
Catalytic Domain
DNA Mismatch Repair
DNA
Substrates
DNA-(Apurinic or Apyrimidinic Site) Lyase
DNA Glycosylases
DNA Repair Enzymes
Repair
Proteolysis
Substrate Specificity
Trypsin
Proteins
Nucleotides
Functional groups
Amino Acids
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cloning, overexpression, and biochemical characterization of the catalytic domain of MutY. / Manuel, Raymond C.; Lloyd, Robert (Stephen).

In: Biochemistry, Vol. 36, No. 37, 16.09.1997, p. 11140-11152.

Research output: Contribution to journalArticle

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