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Detection of OG:A Lesion Mispairs by MutY Relies on a Single His Residue and the 2-Amino Group of 8-Oxoguanine.

ABSTRACT: MutY glycosylase excises adenines misincorporated opposite the oxidatively damaged lesion, 8-oxo-7,8-dihydroguanine (OG), to initiate base excision repair and prevent G to T transversion mutations. Successful repair requires MutY recognition of the OG:A mispair amidst highly abundant and structurally similar undamaged DNA base pairs. Herein we use a combination of in vitro and bacterial cell repair assays with single-molecule fluorescence microscopy to demonstrate that both a C-terminal domain histidine residue and the 2-amino group of OG base are critical for MutY detection of OG:A sites. These studies are the first to directly link deficiencies in MutY lesion detection with incomplete cellular repair. These results suggest that defects in lesion detection of human MutY (MUTYH) variants may prove predictive of early-onset colorectal cancer known an MUTYH-associated polyposis. Furthermore, unveiling these specific molecular determinants for repair makes it possible to envision new MUTYH-specific cancer therapies.

SUBMITTER: Lee AJ

PROVIDER: S-EPMC7593828 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Structural Basis for Finding OG Lesions and Avoiding Undamaged G by the DNA Glycosylase MutY.

Project description:The adenine glycosylase MutY selectively initiates repair of OG:A lesions and, by comparison, avoids G:A mispairs. The ability to distinguish these closely related substrates relies on the C-terminal domain of MutY, which structurally resembles MutT. To understand the mechanism for substrate specificity, we crystallized MutY in complex with DNA containing G across from the high-affinity azaribose transition state analogue. Our structure shows that G is accommodated by the OG site and highlights the role of a serine residue in OG versus G discrimination. The functional significance of Ser308 and its neighboring residues was evaluated by mutational analysis, revealing the critical importance of a β loop in the C-terminal domain for mutation suppression in cells, and biochemical performance in vitro. This loop comprising residues Phe307, Ser308, and His309 (Geobacillus stearothermophilus sequence positions) is conserved in MutY but absent in MutT and other DNA repair enzymes and may therefore serve as a MutY-specific target exploitable by chemical biological probes.

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MutY, an adenine glycosylase active on G-A mispairs, has homology to endonuclease III.

Project description:The mutY gene of Escherichia coli, which codes for an adenine glycosylase that excises the adenine of a G-A mispair, has been cloned and sequenced. The mutY gene codes for a protein of 350 amino acids (Mr = 39,123) and the clone genetically complements the mutY strain. The protein shows significant sequence homology to E. coli endonuclease III, an enzyme that has previously been shown to have glycosylase activity on damaged base pairs. Sequence analysis suggests that, like endonuclease III, MutY is an iron-sulfur protein with a [4Fe-4S]2+ cluster.

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8-oxoguanine DNA glycosylases: one lesion, three subfamilies.

Project description:Amongst the four bases that form DNA, guanine is the most susceptible to oxidation, and its oxidation product, 7,8-dihydro-8-oxoguanine (8-oxoG) is the most prevalent base lesion found in DNA. Fortunately, throughout evolution cells have developed repair mechanisms, such as the 8-oxoguanine DNA glycosylases (OGG), which recognize and excise 8-oxoG from DNA thereby preventing the accumulation of deleterious mutations. OGG are divided into three subfamilies, OGG1, OGG2 and AGOG, which are all involved in the base excision repair (BER) pathway. The published structures of OGG1 and AGOG, as well as the recent availability of OGG2 structures in both apo- and liganded forms, provide an excellent opportunity to compare the structural and functional properties of the three OGG subfamilies. Among the observed differences, the three-dimensional fold varies considerably between OGG1 and OGG2 members, as the latter lack the A-domain involved in 8-oxoG binding. In addition, all three OGG subfamilies bind 8-oxoG in a different manner even though the crucial interaction between the enzyme and the protonated N7 of 8-oxoG is conserved. Finally, the three OGG subfamilies differ with respect to DNA binding properties, helix-hairpin-helix motifs, and specificity for the opposite base.

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Unnatural substrates reveal the importance of 8-oxoguanine for in vivo mismatch repair by MutY.

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Structure-Activity Relationships Reveal Key Features of 8-Oxoguanine: A Mismatch Detection by the MutY Glycosylase.

Project description:Base excision repair glycosylases locate and remove damaged bases in DNA with remarkable specificity. The MutY glycosylases, unusual for their excision of undamaged adenines mispaired to the oxidized base 8-oxoguanine (OG), must recognize both bases of the mispair in order to prevent promutagenic activity. Moreover, MutY must effectively find OG:A mismatches within the context of highly abundant and structurally similar T:A base pairs. Very little is known about the factors that initiate MutY's interaction with the substrate when it first encounters an intrahelical OG:A mispair, or about the order of recognition checkpoints. Here, we used structure-activity relationships (SAR) to investigate the features that influence the in vitro measured parameters of mismatch affinity and adenine base excision efficiency by E. coli MutY. We also evaluated the impacts of the same substrate alterations on MutY-mediated repair in a cellular context. Our results show that MutY relies strongly on the presence of the OG base and recognizes multiple structural features at different stages of recognition and catalysis to ensure that only inappropriately mispaired adenines are excised. Notably, some OG modifications resulted in more dramatic reductions in cellular repair than in the in vitro kinetic parameters, indicating their importance for initial recognition events needed to locate the mismatch within DNA. Indeed, the initial encounter of MutY with its target base pair may rely on specific interactions with the 2-amino group of OG in the major groove, a feature that distinguishes OG:A from T:A base pairs. These results furthermore suggest that inefficient substrate location in human MutY homologue variants may prove predictive for the early onset colorectal cancer phenotype known as MUTYH-Associated Polyposis, or MAP.

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Structural Basis for the Lesion-scanning Mechanism of the MutY DNA Glycosylase.

Project description:The highly mutagenic A:8-oxoguanine (oxoG) base pair is generated mainly by misreplication of the C:oxoG base pair, the oxidation product of the C:G base pair. The A:oxoG base pair is particularly insidious because neither base in it carries faithful information to direct the repair of the other. The bacterial MutY (MUTYH in humans) adenine DNA glycosylase is able to initiate the repair of A:oxoG by selectively cleaving the A base from the A:oxoG base pair. The difference between faithful repair and wreaking mutagenic havoc on the genome lies in the accurate discrimination between two structurally similar base pairs: A:oxoG and A:T. Here we present two crystal structures of the MutY N-terminal domain in complex with either undamaged DNA or DNA containing an intrahelical lesion. These structures have captured for the first time a DNA glycosylase scanning the genome for a damaged base in the very first stage of lesion recognition and the base extrusion pathway. The mode of interaction observed here has suggested a common lesion-scanning mechanism across the entire helix-hairpin-helix superfamily to which MutY belongs. In addition, small angle X-ray scattering studies together with accompanying biochemical assays have suggested a possible role played by the C-terminal oxoG-recognition domain of MutY in lesion scanning.

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Ser 524 is a phosphorylation site in MUTYH and Ser 524 mutations alter 8-oxoguanine (OG): a mismatch recognition.

Project description:MUTYH-associated polyposis (MAP) is a colorectal cancer predisposition syndrome that is caused by inherited biallelic mutations in the base excision repair (BER) gene, MUTYH. MUTYH is a DNA glycosylase that removes adenine (A) misinserted opposite 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG). In this work, wild type (WT) MUTYH overexpressed using a baculovirus-driven insect cell expression system (BEVS) provided significantly higher levels of enzyme compared to bacterial overexpression. The isolated MUTYH enzyme was analyzed for potential post-translational modifications using mass spectrometry. An in vivo phosphorylation site was validated at Serine 524, which is located in the C-terminal OG recognition domain within the proliferating cell nuclear antigen (PCNA) binding region. Characterization of the phosphomimetic (S524D) and phosphoablating (S524A) mutants together with the observation that Ser 524 can be phosphorylated suggest that this residue may play an important regulatory role in vivo by altering stability and OG:A mismatch affinity.

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Differential subcellular localization of human MutY homolog (hMYH) and the functional activity of adenine:8-oxoguanine DNA glycosylase.

Project description:The post-replicative adenine:8-oxo-7,8-dihydroguanine (GO) mismatch is crucial for G:C to T:A transversion. This mismatch is corrected by Escherichia coli MutY which excises the adenine from A:GO. A candidate gene coding for the human counterpart of MutY has been cloned as hMYH. However, the function and enzyme activities of the gene product have not been identified. We previously demonstrated that an epitope-tagged hMYH protein behaves as a mitochondrial protein. In the present study, we have identified an alternative hMYH transcript, termed type 2, which differs in the exon 1 sequence of the known transcript (type 1). A nuclear localization for the type 2 protein was revealed by detection of epitope-tagged protein in COS-7 cells. Expression of both type 1 and type 2 transcripts was reduced in post-mitotic tissues. hMYH cDNA suppressed the mutator phenotype of E.coli mutY. In vitro expressed hMYH showed adenine DNA glycosylase activity toward the A:GO substrate. The protein can bind to A:GO, and to T:GO and G:GO without apparent catalysis. These results represent the first demonstration of the function of the hMYH gene product which is differentially transported into the nucleus or the mitochondria by alternative splicing

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DNA polymerase minor groove interactions modulate mutagenic bypass of a templating 8-oxoguanine lesion.

Project description:A major base lesion resulting from oxidative stress is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG) that has ambiguous coding potential. Error-free DNA synthesis involves 8-oxoG adopting an anti-conformation to base pair with cytosine whereas mutagenic bypass involves 8-oxoG adopting a syn-conformation to base pair with adenine. Left unrepaired the syn-8-oxoG/dAMP base pair results in a G-C to T-A transversion. During base excision repair of this mispair, DNA polymerase (pol) ? is confronted with gap filling opposite 8-oxoG. To determine how pol ? discriminates between anti- and syn-8-oxoG, we introduced a point mutation (R283K) to alter insertion specificity. Kinetic studies demonstrate that this substitution results in an increased fidelity opposite 8-oxoG. Structural studies with R283K pol ? show that the binary DNA complex has 8-oxoG in equilibrium between anti- and syn-forms. Ternary complexes with incoming dCTP resemble the wild-type enzyme, with templating anti-8-oxoG base pairing with incoming cytosine. In contrast to wild-type pol ?, the ternary complex of the R283K mutant with an incoming dATP-analogue and templating 8-oxoG resembles a G-A mismatched structure with 8-oxoG adopting an anti-conformation. These results demonstrate that the incoming nucleotide is unable to induce a syn-8-oxoG conformation without minor groove DNA polymerase interactions that influence templating (anti-/syn-equilibrium) of 8-oxoG while modulating fidelity.

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The trajectory of intrahelical lesion recognition and extrusion by the human 8-oxoguanine DNA glycosylase.

Project description:Efficient search for DNA damage embedded in vast expanses of the DNA genome presents one of the greatest challenges to DNA repair enzymes. We report here crystal structures of human 8-oxoguanine (oxoG) DNA glycosylase, hOGG1, that interact with the DNA containing the damaged base oxoG and the normal base G while they are nested in the DNA helical stack. The structures reveal that hOGG1 engages the DNA using different protein-DNA contacts from those observed in the previously determined lesion recognition complex and other hOGG1-DNA complexes. By applying molecular dynamics simulations, we have determined the pathways taken by the lesion and normal bases when extruded from the DNA helix and their associated free energy profiles. These results reveal how the human oxoG DNA glycosylase hOGG1 locates the lesions inside the DNA helix and facilitates their extrusion for repair.

| S-EPMC7477556 | biostudies-literature

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