Identification of the Structural and Functional Domains of MutY, an DNA Mismatch Repair Enzyme

Abstract

The linear amino acid sequences of the Escherichia coli DNA repair proteins, MutY and endonuclease III, show significant homology, even though these enzymes recognize entirely different substrates. In this study, proteolysis and molecular modeling of MutY were used to elucidate its domain organization. Proteolysis by trypsin cleaved the enzyme into 26-and 13-kDa fragments. NH 2 -terminal sequencing showed that the p13 domain begins at Gln 226 , indicating that the COOH-terminal portion of MutY, absent in endonuclease III, is organized as a separate domain. The large p26 domain is almost equivalent to the size of endonuclease III. Binding activity of the p26 domain to a DNA substrate containing an A⅐G mismatch was comparable with that of the intact enzyme. In vitro studies show that the p26 domain retains adenine glycosylase and AP lyase activity on DNA containing undamaged adenine opposite guanine or 8-oxo-7,8-dihydro-2-deoxyguanine. Although the activity was somewhat reduced, the above results show that the critical amino acid residues involved in substrate binding and catalysis are present in this domain. The structure predicted by molecular modeling indicates that the region of MutY (Met 1 -Trp 216), which is homologous to endonuclease III exhibits a two domain structure, even though this portion is resistant to proteolysis by trypsin.Proteins are constructed on a modular basis, and frequently these modules or domains are known to have unique functions. Isolation and characterization of these domains provide significant insight into the relationship between particular structural elements of the enzyme and its various activities. The mutY gene of Escherichia coli encodes a 39.1-kDa DNA mismatch repair protein. A significant portion of this protein is homologous to the 26.3-kDa E. coli endonuclease III. These two proteins are 66.3% similar and 23.8% identical over a 181-amino acid region (1). Another enzyme with sequence similarity to MutY is the product of the pdg gene in Micrococcus luteus, which recognizes and incises DNA containing cyclobutane pyrimidine dimers (2). The three enzymes mentioned above contain a [4Fe-4S] 2ϩ cluster, coordinated by four cysteine residues which are perfectly conserved in all three proteins.In contrast to their structural similarities, the functional properties of the above three DNA repair proteins are very different. MutY recognizes and removes the undamaged adenine mispaired with guanine, cytosine, 8-oxo-7,8-dihydro-2Ј-deoxyguanine (8-oxo-dG) 1 and 8-oxo-7,8-dihydro-2Ј-deoxyadenine (3) and is also reported to have AP endonuclease activity (4, 5). In vitro experiments show that MutY also recognizes and removes adenine analogs when they are paired with guanine (5).2 Endonuclease III primarily removes oxidized pyrimidines from DNA (6), and the pdg gene product in M. luteus repairs UV-induced pyrimidine dimer lesions in DNA (2). Thus, despite their structural similarities, these DNA repair enzymes have different substrate specificity. The catalytic mechanism of endonuclease III in...

DNA glycosylases in the base excision repair of DNA

“…Interestingly, the 26 kDa fragment retains normal DNA binding and adenine-DNA glycosylase\β-lyase activity against G\A, whereas the activity is dramatically decreased against 8-oxoG\A [101]. Homology modelling has indicated that the 26 kDa proteolytic fragment of MutY has the same overall conformation as EndoIII [102], and structural similarity is consistent with preliminary crystallographic studies (Y. Guan and J. A. Tainer, personal communication).…”

DNA repair glycosylases with a [4Fe–4S] cluster: A redox cofactor for DNA-mediated charge transport?

“…MutY, structurally similar to Endo III [18][19][20][21], is another BER glycosylase that contains a [4Fe-4S] cluster [20]. However, MutY instead removes adenine from 8-oxo-guanine:adenine mispairs [22][23][24][25][26][27][28][29][30][31][32][33][34].…”

A Dimeric Mechanism for Contextual Target Recognition by MutY Glycosylase

“…The prevalent oxidatively damaged base, 8-oxo-guanine, is excised from DNA when base-paired to cytosine by MutM (16 -20) and its eukaryotic homolog, OGG1 (21)(22)(23)(24)(25)(26). Misincorporation of adenines by DNA polymerase opposite 8-oxo-guanines that escape MutM-mediated removal are subsequently repaired by the adenine glycosylase, MutY (20,(27)(28)(29)(30)(31)(32)(33), and its homolog, MYH (16, 34 -36). Additionally, MutT (16,20,(37)(38)(39)(40), a nucleoside triphosphate pyrophosphohydrolase, removes oxidatively damaged dGTPs, thereby preventing their incorporation into DNA.…”