Escherichia coli MutY protein has both N-glycosylase and apurinic/apyrimidinic endonuclease activities on A.C and A.G mispairs.

Tsai-Wu, Jyy-Jih; Liu, Hsin-Fei; Lu, A-Lien

doi:10.1073/pnas.89.18.8779

Cited by 141 publications

(147 citation statements)

References 37 publications

(46 reference statements)

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“…However, there have also been several studies that show MutY to have adenine glycosylase and 3Ј AP endonuclease activities (4,5,28). In a recent report, a putative mammalian homolog of E. coli MutY mismatch repair protein has been shown to recognize similar substrates with the nicking activity observed at the phosphodiester bond 3Ј to the AP site following adenine glycosylase activity (29).…”

Section: Discussionmentioning

confidence: 99%

“…In MutY, a potential role for the Fe-S cluster has been suggested in previous studies which show that renaturation of denatured enzyme requires iron and sulfur to recover the catalytic activity (4). The retention of specific DNA binding activity by the p26 domain indicates that the structural integrity of the [4Fe-4S] 2ϩ cluster coordinated by four cysteine residues is conserved, thereby providing a surface to interact with DNA, and this region may even serve as a portion of the binding pocket.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Manuel

Czerwinski

Lloyd

1996

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Manuel

Czerwinski

Lloyd

1996

Journal of Biological Chemistry

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“…The mutator locus was designated mutY, and the repair activity was found to be independent of the methylation state of the DNA [94,95]. mutY encodes a DNA glycosylase of 36 kDa which, in addition to excision of A opposite G and C, also removes A opposite the oxidized purines 8-oxoG and 7,8-dihydro-8-oxoadenine (8-oxoA) [96]. If 8-oxoG is not removed prior to replication, a C or an A is inserted opposite it.…”

Section: G/t(u)-mismatch Dna Glycosylasesmentioning

confidence: 99%

DNA glycosylases in the base excision repair of DNA

Krokan

Standal

Slupphaug

1997

Biochemical Journal

766

566

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A wide range of cytotoxic and mutagenic DNA bases are removed by different DNA glycosylases, which initiate the base excision repair pathway. DNA glycosylases cleave the Nglycosylic bond between the target base and deoxyribose, thus releasing a free base and leaving an apurinic\apyrimidinic (AP) site. In addition, several DNA glycosylases are bifunctional, since they also display a lyase activity that cleaves the phosphodiester backbone 3h to the AP site generated by the glycosylase activity. Structural data and sequence comparisons have identified common features among many of the DNA glycosylases. Their active sites have a structure that can only bind extrahelical target bases, as observed in the crystal structure of human uracil-DNA glycosylase in a complex with double-stranded DNA. Nucleotide flipping is apparently actively facilitated by the enzyme. With bacteriophage T4 endonuclease V, a pyrimidine-

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“…DNA Glycosylase Assay-The DNA cleavage activity (DNA glycosylase activity followed by heating) of hMYH was assayed similarly as E. coli MutY glycosylase as described (46,50,51) except different buffer and incubation times were used. The DNA substrate is a 20-mer duplex DNA containing an A/8-oxoG mismatch (5Ј-CCGAGGAATTAGC-CCCCTTCTGC-3Ј/3Ј-GGCTCCTTAAOCGGGGGAAGACG5Ј, where O represents 8-oxoG) that is labeled at the 3Ј-end of the mismatched A-containing strand.…”

Section: Methodsmentioning

confidence: 99%

Human Homolog of the MutY Repair Protein (hMYH) Physically Interacts with Proteins Involved in Long Patch DNA Base Excision Repair

Parker¹,

Gu²,

Mahoney

et al. 2001

Journal of Biological Chemistry

200

207

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The human MutY homolog (hMYH) is a DNA glycosylase involved in the removal of adenines or 2-hydroxyadenines misincorporated with template guanines or 7,8-dihydro-8-oxodeoxyguanines. hMYH is associated in vivo with apurinic/apyrimidinic endonuclease (APE1), proliferating cell nuclear antigen (PCNA), and replication protein A (RPA) in HeLa nuclear extracts as shown by immunoprecipitation and Western blotting. However, binding of hMYH to DNA polymerases ␤ and ␦ was not detected. By using constructs containing different portions of hMYH fused to glutathione S-transferase, we have demonstrated that the APE1-binding site is at a region around amino acid residue 300, that the PCNA binding activity is located at the C terminus, and that RPA binds to the N terminus of hMYH. A peptide consisting of residues 505-527 of hMYH that contains a conserved PCNA-binding motif binds PCNA, and subsequent amino acid substitution identified Phe-518 and Phe-519 as essential residues required for PCNA binding. RPA binds to a peptide that consists of residues 6 -32 of hMYH and contains a conserved RPA-binding motif. The PCNA-and RPA-binding sites of hMYH are further confirmed by peptide and antibody titration. These results suggest that hMYH repair is a long patch base excision repair pathway.

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Escherichia coli MutY protein has both N-glycosylase and apurinic/apyrimidinic endonuclease activities on A.C and A.G mispairs.

Cited by 141 publications

References 37 publications

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

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

DNA glycosylases in the base excision repair of DNA

Human Homolog of the MutY Repair Protein (hMYH) Physically Interacts with Proteins Involved in Long Patch DNA Base Excision Repair

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