The Experts below are selected from a list of 3159 Experts worldwide ranked by ideXlab platform
Sheila S. David - One of the best experts on this subject based on the ideXlab platform.
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Designer Fluorescent Adenines Enable Real-Time Monitoring of MUTYH Activity.
ACS central science, 2020Co-Authors: Ru-yi Zhu, Chandrima Majumdar, Cindy Khuu, Mariarosaria De Rosa, Patricia L. Opresko, Sheila S. David, Eric T. KoolAbstract:The human DNA base excision repair enzyme MUTYH (MutY homolog DNA glycosylase) excises undamaged adenine that has been misincorporated opposite the oxidatively damaged 8-oxoG, preventing transversion mutations and serving as an important defense against the deleterious effects of this damage. Mutations in the MUTYH gene predispose patients to MUTYH-associated polyposis and colorectal cancer, and MUTYH expression has been documented as a biomarker for pancreatic cancer. Measuring MUTYH activity is therefore critical for evaluating and diagnosing disease states as well as for testing this enzyme as a potential therapeutic target. However, current methods for measuring MUTYH activity rely on indirect electrophoresis and radioactivity assays, which are difficult to implement in biological and clinical settings. Herein, we synthesize and identify novel fluorescent adenine derivatives that can act as direct substrates for excision by MUTYH as well as bacterial MutY. When incorporated into synthetic DNAs, the resulting fluorescently modified adenine-release turn-on (FMART) probes report on enzymatic base excision activity in real time, both in vitro and in mammalian cells and human blood. We also employ the probes to identify several promising small-molecule modulators of MUTYH by employing FMART probes for in vitro screening.
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When you're strange: Unusual features of the MUTYH glycosylase and implications in cancer.
DNA repair, 2019Co-Authors: Alan G Raetz, Sheila S. DavidAbstract:MUTYH is a base-excision repair glycosylase that removes adenine opposite 8-oxoguanine (OG). Variants of MUTYH defective in functional activity lead to MUTYH-associated polyposis (MAP), which progresses to cancer with very high penetrance. Whole genome and whole exome sequencing studies have found MUTYH deficiencies in an increasing number of cancer types. While the canonical OG:A repair activity of MUTYH is well characterized and similar to bacterial MutY, here we review more recent evidence that MUTYH has activities independent of OG:A repair and appear centered on the interdomain connector (IDC) region of MUTYH. We summarize evidence that MUTYH is involved in rapid DNA damage response (DDR) signaling, including PARP activation, 9-1-1 and ATR signaling, and SIRT6 activity. MUTYH alters survival and DDR to a wide variety of DNA damaging agents in a time course that is not consistent with the formation of OG:A mispairs. Studies that suggest MUTYH inhibits the repair of alkyl-DNA damage and cyclopyrimidine dimers (CPDs) is reviewed, and evidence of a synthetic lethal interaction with mismatch repair (MMR) is summarized. Based on these studies we suggest that MUTYH has evolved from an OG:A mispair glycosylase to a multifunctional scaffold for DNA damage response signaling.
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cellular assays for studying the fe s cluster containing base excision repair glycosylase MUTYH and homologs
Methods in Enzymology, 2018Co-Authors: Chandrima Majumdar, Alan G Raetz, Cindy Khuu, Nicole N Nunez, Sheila S. DavidAbstract:Abstract Many DNA repair enzymes, including the human adenine glycosylase MUTYH, require iron–sulfur (Fe–S) cluster cofactors for DNA damage recognition and subsequent repair. MUTYH prokaryotic and eukaryotic homologs are a family of adenine (A) glycosylases that cleave A when mispaired with the oxidatively damaged guanine lesion, 8-oxo-7,8-dihydroguanine (OG). Faulty OG:A repair has been linked to the inheritance of missense mutations in the MUTYH gene. These inherited mutations can result in the onset of a familial colorectal cancer disorder known as MUTYH-associated polyposis (MAP). While in vitro studies can be exceptional at unraveling how MutY interacts with its OG:A substrate, cell-based assays are needed to provide a cellular context to these studies. In addition, strategic comparison of in vitro and in vivo studies can provide exquisite insight into the search, selection, excision process, and the coordination with protein partners, required to mediate full repair of the lesion. A commonly used assay is the rifampicin resistance assay that provides an indirect evaluation of the intrinsic mutation rate in Escherichia coli (E. coli or Ec), read out as antibiotic-resistant cell growth. Our laboratory has also developed a bacterial plasmid-based assay that allows for direct evaluation of repair of a defined OG:A mispair. This assay provides a means to assess the impact of catalytic defects in affinity and excision on overall repair. Finally, a mammalian GFP-based reporter assay has been developed that more accurately models features of mammalian cells. Taken together, these assays provide a cellular context to the repair activity of MUTYH and its homologs that illuminates the role these enzymes play in preventing mutations and disease.
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Repair of 8-oxoG:A mismatches by the MUTYH glycosylase: Mechanism, metals and medicine.
Free radical biology & medicine, 2017Co-Authors: Douglas M Banda, Nicole N Nunez, Michael A. Burnside, Katie M. Bradshaw, Sheila S. DavidAbstract:Reactive oxygen and nitrogen species (RONS) may infringe on the passing of pristine genetic information by inducing DNA inter- and intra-strand crosslinks, protein-DNA crosslinks, and chemical alterations to the sugar or base moieties of DNA. 8-Oxo-7,8-dihydroguanine (8-oxoG) is one of the most prevalent DNA lesions formed by RONS and is repaired through the base excision repair (BER) pathway involving the DNA repair glycosylases OGG1 and MUTYH in eukaryotes. MUTYH removes adenine (A) from 8-oxoG:A mispairs, thus mitigating the potential of G:C to T:A transversion mutations from occurring in the genome. The paramount role of MUTYH in guarding the genome is well established in the etiology of a colorectal cancer predisposition syndrome involving variants of MUTYH, referred to as MUTYH-associated polyposis (MAP). In this review, we highlight recent advances in understanding how MUTYH structure and related function participate in the manifestation of human disease such as MAP. Here we focus on the importance of MUTYH's metal cofactor sites, including a recently discovered "Zinc linchpin" motif, as well as updates to the catalytic mechanism. Finally, we touch on the insight gleaned from studies with MAP-associated MUTYH variants and recent advances in understanding the multifaceted roles of MUTYH in the cell, both in the prevention of mutagenesis and tumorigenesis.
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Distinct functional consequences of MUTYH variants associated with colorectal cancer: Damaged DNA affinity, glycosylase activity and interaction with PCNA and Hus1.
DNA repair, 2015Co-Authors: Megan K Brinkmeyer, Sheila S. DavidAbstract:MUTYH is a base excision repair (BER) enzyme that prevents mutations in DNA associated with 8-oxoguanine (OG) by catalyzing the removal of adenine from inappropriately formed OG:A base-pairs. Germline mutations in the MUTYH gene are linked to colorectal polyposis and a high risk of colorectal cancer, a syndrome referred to as MUTYH-associated polyposis (MAP). There are over 300 different MUTYH mutations associated with MAP and a large fraction of these gene changes code for missense MUTYH variants. Herein, the adenine glycosylase activity, mismatch recognition properties, and interaction with relevant protein partners of human MUTYH and five MAP variants (R295C, P281L, Q324H, P502L, and R520Q) were examined. P281L MUTYH was found to be severely compromised both in DNA binding and base excision activity, consistent with the location of this variation in the iron-sulfur cluster (FCL) DNA binding motif of MUTYH. Both R295C and R520Q MUTYH were found to have low fractions of active enzyme, compromised affinity for damaged DNA, and reduced rates for adenine excision. In contrast, both Q324H and P502L MUTYH function relatively similarly to WT MUTYH in both binding and glycosylase assays. However, P502L and R520Q exhibited reduced affinity for PCNA (proliferation cell nuclear antigen), consistent with their location in the PCNA-binding motif of MUTYH. Whereas, only Q324H, and not R295C, was found to have reduced affinity for Hus1 of the Rad9-Hus1-Rad1 complex, despite both being localized to the same region implicated for interaction with Hus1. These results underscore the diversity of functional consequences due to MUTYH variants that may impact the progression of MAP.
Yusaku Nakabeppu - One of the best experts on this subject based on the ideXlab platform.
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MUTYH Deficiency Is Associated with Attenuated Pulmonary Fibrosis in a Bleomycin-Induced Model.
Oxidative medicine and cellular longevity, 2020Co-Authors: Qingmin Sun, Yusaku Nakabeppu, Jingwen Chen, Jiaqi Kang, Yan Ren, Yaping WangAbstract:Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible lung disease of unknown etiology with limited survival. IPF incidence and prevalence increase significantly with aging, which is associated with an age-related accumulation of oxidative DNA damage. The MUTYH gene is involved in the base excision repair (BER) system, which is critical for repairing the misincorporated adenine that is opposite to the oxidized guanine base, 8-oxoguanine, and maintaining the fidelity of DNA replication. We used MUTYH knockout mice and a bleomycin-induced pulmonary fibrosis model to test the effect of MUTYH deficiency on lesion progression. Unexpectedly, a much less severe lesion of pulmonary fibrosis was observed in MUTYH -/- than in MUTYH +/+ mice, which was supported by assay on protein levels of TGF-β1 and both fibrotic markers, α-SMA and Vimentin, in pulmonary tissues of the model animals. Mechanically, MUTYH deficiency prevented the genomic DNA of pulmonary tissue cells from the buildup of single-strand breaks (SSBs) of DNA and maintained the integrity of mtDNA. Furthermore, increased mitochondrial dynamic regulation and mitophagy were detected in pulmonary tissues of the bleomycin-induced MUTYH -/- model mice, which could reduce the pulmonary epithelial cell apoptosis. Our results suggested that MUTYH deficiency could even induce protective responses of pulmonary tissue under severe oxidative stress.
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Oxidative stress induces different tissue dependent effects on MUTYH-deficient mice.
Free radical biology & medicine, 2019Co-Authors: Jingwen Chen, Zhenqian Huang, Jiaqi Kang, Yan Ren, Wei Gao, Jingmei Wang, Weidong Ding, Yusaku NakabeppuAbstract:8-oxoguanine (8-oxoG) is one of the most prevalent genotoxic lesions, and it is generated in DNA attacked by reactive oxygen species (ROS). Adenine misincorporated opposite to 8-oxoG during replication is excised by MutY homolog (MUTYH), an important protein of the base excision repair (BER) system. MUTYH plays an important role in the maintenance of genomic integrity, but the functional consequences of MUTYH deficiency are not fully understood. In the current study, we investigated the histological and functional changes of five tissues (hippocampus, heart, liver, kidney and lung) and their molecular basis in MUTYH-/- and wild-type mice exposed to D-galactose (D-gal). Our data indicated that MUTYH deficiency hindered the weight gain of experimental mice and induced substantial alterations of 8-oxoG content and superoxide dismutase (SOD) activity, but no significant histological and functional impairment appeared in the investigated tissues of MUTYH- deficient mice without D-gal exposure. Under low-dose D-gal exposure, MUTYH deficiency altered expression of genes involved in mitochondrial unfolded protein response (UPRmt) in the heart, liver and lung, and caused an enhanced expression of mitochondrial dynamics proteins (MDPs) in hippocampus and liver. The stress responses could maintain mitochondrial proteostasis and function. However, such responses were not noted when experiencing excessive damage burden induced by high-dose D-gal exposure, in which MUTYH deficiency increased accumulation of 8-oxoG and aggravated mitonuclear protein imbalance, as well as histological lesions in heart, liver and kidney. A higher sensitivity to ROS-induced cardiotoxicity with high-dose D-gal exposure was noticed in MUTYH-/- mice. However, no differences in learning and memory impairments were observed between MUTYH-/- and wild-type mice with high-dose D-gal exposure. In conclusion, our data demonstrated that MUTYH deficiency has different impacts on various tissues based on the degree of oxidative stress.
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dna glycosylase encoded by MUTYH functions as a molecular switch for programmed cell death under oxidative stress to suppress tumorigenesis
Cancer Science, 2011Co-Authors: Sugako Oka, Yusaku NakabeppuAbstract:8-oxoguanine is a major base lesion in DNA or in nucleotides caused by oxidative stress, and is highly mutagenic because it can pair with adenine as well as cytosine. Adenine DNA glycosylase, encoded by the human mutY homolog gene, MUTYH, excises adenine in the nascent strand when inserted opposite 8-oxoguanine in template DNA, and thus suppresses mutagenesis caused by 8-oxoguanine that has accumulated in DNA due to oxidative stress. Several germ-line mutations in MUTYH are predisposed to MUTYH-associated polyposis, an autosomal recessive disorder characterized by multiple colorectal adenomas and carcinomas. Loss of function of MUTYH leads to an accumulation of somatic mutations in APC and KRAS genes, resulting in the development of adenomas/carcinomas. We recently demonstrated that accumulation of 8-oxoguanine in nuclear and mitochondrial DNA triggers two distinct cell death pathways that are independent of each other. Both pathways are initiated by the accumulation of MUTYH-generated single-strand breaks (SSBs) in nuclear or mitochondrial DNA. Our findings indicate that MUTYH-induced cell death due to oxidative stress results in an efficient elimination of mutagenic cells that have accumulated high levels of 8-oxoguanine in their DNAs. It is most likely that loss of function of MUTYH in stem or progenitor cells in the intestinal epithelium of MUTYH-associated polyposis patients results in escape from programmed cell death; however, accumulated 8-oxoguanine causes various mutations in APC or KRAS genes in these proliferative cells, thereby promoting tumorigenesis. We thus propose that MUTYH suppresses tumorigenesis under conditions of oxidative stress by inducing cell death and by suppressing mutagenesis. (Cancer Sci 2011; 102: 677–682)
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Altered expression of MUTYH and an increase in 8‐hydroxydeoxyguanosine are early events in ulcerative colitis‐associated carcinogenesis
The Journal of pathology, 2009Co-Authors: Masaki Gushima, Yusaku Nakabeppu, Minako Hirahashi, Takayuki Matsumoto, Kouhei Fujita, Ritsuko Fujisawa, Kazuhiro Mizumoto, Mitsuo Iida, Takashi Yao, Masazumi TsuneyoshiAbstract:8-Hydroxy-guanine (8-OH-G) mismatches readily with adenine residues, leading to a G : C to T : A transversion mutation. The human mutY homologue (MUTYH) excises adenine misincorporated opposite 8-OH-G during replication and suppresses mutations caused by reactive oxygen species. We defined the expression of 8-hydroxydeoxyguanosine (8-OHdG) and MUTYH in ulcerative colitis (UC)-associated neoplasia by immunohistochemistry and compared this with expression in UC patients without neoplasia and patients unaffected by UC. We also performed mutation analyses for MUTYH and K-ras. 8-OHdG was expressed more intensely in the mucosa of UC-associated neoplasia and UC without neoplasm than in the mucosa unaffected by UC. Immunohistochemistry with two different types of MUTYH antibody showed that UC-associated neoplasia and UC without neoplasia exhibited strong cytoplasmic expression and attenuated nuclear expression of MUTYH when compared with patients unaffected by UC. No pathological MUTYH mutations were detected in any of the UC-associated neoplasia cases. However, K-ras mutation was detected in two cases, one of which showed G : C to T : A transversion mutation and attenuated nuclear staining of MUTYH. In conclusion, inflamed mucosa of UC is exposed to oxidative damage. An increase in cytoplasmic MUTYH, rather than its mutation, may contribute to the promotion of carcinogenesis in UC. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Genomic and functional analyses of MUTYH in Japanese patients with adenomatous polyposis.
Clinical genetics, 2008Co-Authors: Ritsuko Yanaru-fujisawa, Yasuhiro Ushijima, Yusaku Nakabeppu, Masaki Gushima, Minako Hirahashi, Takayuki Matsumoto, Motohiro Esaki, Tsuneyoshi Yao, M. IidaAbstract:The present study was undertaken to elucidate germ line mutations of the base excision repair gene, MUTYH, in Japanese patients with adenomatous polyposis. We screened germ line mutations of adenomatous polyposis coli (APC) gene and MUTYH in 66 Japanese patients with adenomatous polyposis. APC was screened by the protein truncation test, while MUTYH was screened by polymerase chain reaction-based single-strand conformation polymorphism and direct sequencing. The nicking assay was applied in order to evaluate the DNA glycosylase activity of the identified MUTYH variant. In this study, Seven MUTYH variants were identified in 16 of 21 APC-negative patients. Q324H mutation was the most frequent mutation, with an allele frequency of 49%. Two patients carried biallelic mutations other than Q324H; a patient had biallelic G272E and A359V mutations, while the other had compound heterozygotes of P18L and G25D mutations. Nicking assay for G272E using the corresponding mouse MUTYH mutant with G257E revealed that G272E is a variant to cause an impaired DNA glycosylase activity. Homozygous MUTYH mutation accounts for approximately 10% of Japanese patients with adenomatous polyposis. G272E may be one of the mutations specific to patients with adenomatous polyposis in East Asia.
Nicole N Nunez - One of the best experts on this subject based on the ideXlab platform.
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the zinc linchpin motif in the dna repair glycosylase MUTYH identifying the zn2 ligands and roles in damage recognition and repair
Journal of the American Chemical Society, 2018Co-Authors: Nicole N Nunez, Anisha N Rajavel, Cindy Khuu, Satheesan C Babu, Steve J Bertolani, Jensen M Spear, Jeremy A Armas, Jon D Wright, Justin B Siegel, Carmay LimAbstract:The DNA base excision repair (BER) glycosylase MUTYH prevents DNA mutations by catalyzing adenine (A) excision from inappropriately formed 8-oxoguanine (8-oxoG):A mismatches. The importance of this mutation suppression activity in tumor suppressor genes is underscored by the association of inherited variants of MUTYH with colorectal polyposis in a hereditary colorectal cancer syndrome known as MUTYH-associated polyposis, or MAP. Many of the MAP variants encompass amino acid changes that occur at positions surrounding the two-metal cofactor-binding sites of MUTYH. One of these cofactors, found in nearly all MUTYH orthologs, is a [4Fe–4S]2+ cluster coordinated by four Cys residues located in the N-terminal catalytic domain. We recently uncovered a second functionally relevant metal cofactor site present only in higher eukaryotic MUTYH orthologs: a Zn2+ ion coordinated by three Cys residues located within the extended interdomain connector (IDC) region of MUTYH that connects the N-terminal adenine excision a...
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cellular assays for studying the fe s cluster containing base excision repair glycosylase MUTYH and homologs
Methods in Enzymology, 2018Co-Authors: Chandrima Majumdar, Alan G Raetz, Cindy Khuu, Nicole N Nunez, Sheila S. DavidAbstract:Abstract Many DNA repair enzymes, including the human adenine glycosylase MUTYH, require iron–sulfur (Fe–S) cluster cofactors for DNA damage recognition and subsequent repair. MUTYH prokaryotic and eukaryotic homologs are a family of adenine (A) glycosylases that cleave A when mispaired with the oxidatively damaged guanine lesion, 8-oxo-7,8-dihydroguanine (OG). Faulty OG:A repair has been linked to the inheritance of missense mutations in the MUTYH gene. These inherited mutations can result in the onset of a familial colorectal cancer disorder known as MUTYH-associated polyposis (MAP). While in vitro studies can be exceptional at unraveling how MutY interacts with its OG:A substrate, cell-based assays are needed to provide a cellular context to these studies. In addition, strategic comparison of in vitro and in vivo studies can provide exquisite insight into the search, selection, excision process, and the coordination with protein partners, required to mediate full repair of the lesion. A commonly used assay is the rifampicin resistance assay that provides an indirect evaluation of the intrinsic mutation rate in Escherichia coli (E. coli or Ec), read out as antibiotic-resistant cell growth. Our laboratory has also developed a bacterial plasmid-based assay that allows for direct evaluation of repair of a defined OG:A mispair. This assay provides a means to assess the impact of catalytic defects in affinity and excision on overall repair. Finally, a mammalian GFP-based reporter assay has been developed that more accurately models features of mammalian cells. Taken together, these assays provide a cellular context to the repair activity of MUTYH and its homologs that illuminates the role these enzymes play in preventing mutations and disease.
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Repair of 8-oxoG:A mismatches by the MUTYH glycosylase: Mechanism, metals and medicine.
Free radical biology & medicine, 2017Co-Authors: Douglas M Banda, Nicole N Nunez, Michael A. Burnside, Katie M. Bradshaw, Sheila S. DavidAbstract:Reactive oxygen and nitrogen species (RONS) may infringe on the passing of pristine genetic information by inducing DNA inter- and intra-strand crosslinks, protein-DNA crosslinks, and chemical alterations to the sugar or base moieties of DNA. 8-Oxo-7,8-dihydroguanine (8-oxoG) is one of the most prevalent DNA lesions formed by RONS and is repaired through the base excision repair (BER) pathway involving the DNA repair glycosylases OGG1 and MUTYH in eukaryotes. MUTYH removes adenine (A) from 8-oxoG:A mispairs, thus mitigating the potential of G:C to T:A transversion mutations from occurring in the genome. The paramount role of MUTYH in guarding the genome is well established in the etiology of a colorectal cancer predisposition syndrome involving variants of MUTYH, referred to as MUTYH-associated polyposis (MAP). In this review, we highlight recent advances in understanding how MUTYH structure and related function participate in the manifestation of human disease such as MAP. Here we focus on the importance of MUTYH's metal cofactor sites, including a recently discovered "Zinc linchpin" motif, as well as updates to the catalytic mechanism. Finally, we touch on the insight gleaned from studies with MAP-associated MUTYH variants and recent advances in understanding the multifaceted roles of MUTYH in the cell, both in the prevention of mutagenesis and tumorigenesis.
Margherita Bignami - One of the best experts on this subject based on the ideXlab platform.
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The MUTYH base excision repair gene protects against inflammation-associated colorectal carcinogenesis
Oncotarget, 2015Co-Authors: Francesca Grasso, Margherita Bignami, Gabriele De Luca, Monica Boirivant, Serena Di Meo, Luca Pasquini, Stefania Rossi, Mauro Biffoni, Emma Di CarloAbstract:// Francesca Grasso 1, 2 , Serena Di Meo 3, 4 , Gabriele De Luca 5 , Luca Pasquini 5 , Stefania Rossi 5 , Monica Boirivant 6 , Mauro Biffoni 5 , Margherita Bignami 1 , Emma Di Carlo 3, 4 1 Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome, Italy 2 Department of Science, University Roma Tre, Rome, Italy 3 Ce.S.I. Biotech, Aging Research Center, “G. d’Annunzio” University Foundation, Chieti, Italy 4 Pathological Anatomy and Molecular Medicine, Department of Medicine and Sciences of Aging, “G. d’Annunzio” University, Chieti, Italy 5 Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita, Rome, Italy 6 Department of Infectious Parasitic and Immuno-mediated Diseases, Istituto Superiore di Sanita, Rome, Italy Correspondence to: Margherita Bignami, e-mail: margherita.bignami@gmail.com Keywords: MUTYH, azoxymethane, DSS, colorectal cancer, inflammation Received: May 05, 2015 Accepted: June 05, 2015 Published: June 18, 2015 We dedicate this paper to the late Prof. Piero Musiani, whose enthusiasm for science was a continuous inspiration to us. ABSTRACT MUTYH DNA glycosylase removes mismatched adenine opposite 7, 8-dihydro-8-oxoguanine (8-oxoG), which is the major mutagenic lesion induced by oxidative stress. Biallelic mutations in MUTYH are associated with MUTYH-Associated polyposis (MAP) and increased risk in colorectal cancer (CRC). We investigated cancer susceptibility associated with MUTYH inactivation in a mouse model of inflammation-dependent carcinogenesis induced by azoxymethane (AOM) and dextran sulphate (DSS). MUTYH −/− mice were more sensitive than wild-type (WT) animals to AOM/DSS toxicity and accumulated DNA 8-oxoG in their gastrointestinal tract. AOM/DSS-induced colonic adenomas were significantly more numerous in MUTYH −/− than in WT animals, and frequently showed a tubulo-villous feature along with high-grade dysplasia and larger size lesions. This condition resulted in a greater propensity to develop adenocarcinomas. The colon of untreated MUTYH −/− mice expressed higher basal levels of pro-inflammatory cytokines GM-CSF and IFNγ, and treatment with AOM/DSS induced an early decrease in circulating CD4+ and CD8+ T lymphocytes and an increase in myeloid-derived suppressor cells (MDSCs). Adenomas from MUTYH −/− mice had a greater infiltrate of Foxp3+ T regulatory cells, granulocytes, macrophages, MDSCs and strong expression of TGF-β-latency-associated peptide and IL6. Our findings indicate that MUTYH loss is associated with an increase in CRC risk, which involves immunosuppression and altered inflammatory response. We propose that the AOM/DSS initiation/promotion protocol in MUTYH −/− mice provides a good model for MAP.
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MUTYH mediates the toxicity of combined DNA 6-thioguanine and UVA radiation
Oncotarget, 2014Co-Authors: Francesca Grasso, Vitalba Ruggieri, Gabriele De Luca, Paola Leopardi, Maria Teresa Mancuso, I Casorelli, Pietro Pichierri, Peter Karran, Margherita BignamiAbstract:// Francesca Grasso 1,2,* , Vitalba Ruggieri 3,* , Gabriele De Luca 1 , Paola Leopardi 1 , Maria Teresa Mancuso 4 , Ida Casorelli 5 , Pietro Pichierri 1 , Peter Karran 6 and Margherita Bignami 1 1 Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome, Italy 2 Department of Science, University Roma Tre, Rome, Italy 3 Laboratory of Pre-Clinical and Translational Research, IRCCS, Referral Cancer Center of Basilicata, Rionero in Vulture, Italy 4 Laboratory of Radiation Biology and Biomedicine, Agenzia Nazionale per le Nuove Tecnologie, l’Energia e lo Sviluppo Economico Sostenibile (ENEA) CR-Casaccia, Rome, Italy 5 Department of Immunohematology and Transfusion Unit, Azienda Ospedaliera Sant’Andrea, Rome, Italy 6 Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, UK * These authors contributed equally to this work Correspondence: Margherita Bignami, email: // Keywords : MUTYH, 6-thioguanine, azathioprine, UVA Received : October 22, 2014 Accepted : December 01, 2014 Published : December 02, 2014 Abstract The therapeutic thiopurines, including the immunosuppressant azathioprine (Aza) cause the accumulation of the UVA photosensitizer 6-thioguanine (6-TG) in the DNA of the patients’ cells. DNA 6-TG and UVA are synergistically cytotoxic and their interaction causes oxidative damage. The MUTYH DNA glycosylase participates in the base excision repair of oxidized DNA bases. Using MUTYH -nullmouse fibroblasts (MEFs) we examined whether MUTYH provides protection against the lethal effects of combined DNA 6-TG/UVA. Surprisingly, MUTYH -null MEFs were more resistant than wild-type MEFs, despite accumulating higher levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG).Their enhanced 6-TG/UVA resistance reflected the absence of the MUTYH protein and MEFs expressing enzymatically-dead human variants were as sensitive as wild-type cells. Consistent with their enhanced resistance, MUTYH -null cells sustained fewer DNA strand breaks and lower levels of chromosomal damage after 6-TG/UVA. Although 6-TG/UVA treatment caused early checkpoint activation irrespective of the MUTYH status, M utyh -null cells failed to arrest in S-phase at late time points. MUTYH-dependent toxicity was also apparent in vivo . MUTYH -/- mice survived better than wild-type during a 12-month chronicexposure to Aza/UVA treatments that significantly increased levels of skin DNA 8-oxoG. Two squamous cell skin carcinomas arose in Aza/UVA treated MUTYH -/- mice whereas similarly treated wild-type animals remained tumor-free.
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Understanding the role of the Q338H MUTYH variant in oxidative damage repair
Nucleic acids research, 2013Co-Authors: Eleonora Turco, Margherita Bignami, Maria Teresa Russo, Paolo Degan, Anna Minoprio, Ilenia Ventura, Paola Torreri, Sara Molatore, Guglielmina Nadia Ranzani, Filomena MazzeiAbstract:The MUTYH DNA–glycosylase is indirectly engaged in the repair of the miscoding 7,8-dihydro-8oxo-2 0 -deoxyguanine (8-oxodG) lesion by removing adenine erroneously incorporated opposite the oxidized purine. Inherited biallelic mutations in the MUTYH gene are responsible for a recessive syndrome, the MUTYH-associated polyposis (MAP), which confers an increased risk of colorectal cancer. In this study, we functionally characterized the Q338H variant using recombinant proteins, as well as cell-based assays. This is a common variant among human colorectal cancer genes, which is generally considered, unrelated to the MAP phenotype but recently indicated as a low-penetrance allele. We demonstrate that the Q338H variant retains a wild-type DNA–glycosylase activity in vitro, but it shows a reduced ability to interact with the replication sensor RAD9:RAD1: HUS1 (9–1–1) complex. In comparison with MUTYH � /� mouse embryo fibroblasts expressing a wild-type MUTYH cDNA, the expression of Q338H variant was associated with increased levels of DNA 8-oxodG, hypersensitivity to oxidant and accumulation of the population in the S phase of the cell cycle. Thus, an inefficient interaction of MUTYH with the 9–1–1 complex leads to a repairdefective phenotype, indicating that a proper communication between MUTYH enzymatic function and the S phase checkpoint is needed for effective repair of oxidative damage.
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Role of MUTYH in human cancer.
Mutation Research, 2013Co-Authors: Filomena Mazzei, Alessandra Viel, Margherita BignamiAbstract:MUTYH, a human ortholog of MutY, is a post-replicative DNA glycosylase, highly conserved throughout evolution, involved in the correction of mismatches resulting from a faulty replication of the oxidized base 8-hydroxyguanine (8-oxodG). In particular removal of adenine from A:8-oxodG mispairs by MUTYH activity is followed by error-free base excision repair (BER) events, leading to the formation of C:8-oxodG base pairs. These are the substrate of another BER enzyme, the OGG1 DNA glycosylase, which then removes 8-oxodG from DNA. Thus the combined action of OGG1 and MUTYH prevents oxidative damage-induced mutations, i.e. GC->TA transversions. Germline mutations in MUTYH are associated with a recessively heritable colorectal polyposis, now referred to as MUTYH-associated polyposis (MAP). Here we will review the phenotype(s) associated with MUTYH inactivation from bacteria to mammals, the structure of the MUTYH protein, the molecular mechanisms of its enzymatic activity and the functional characterization of MUTYH variants. The relevance of these results will be discussed to define the role of specific human mutations in colorectal cancer risk together with the possible role of MUTYH inactivation in sporadic cancer.
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MUTYH mutations associated with familial adenomatous polyposis functional characterization by a mammalian cell based assay
Human Mutation, 2010Co-Authors: Sara Molatore, Filomena Mazzei, Maria Teresa Russo, Flavia Barone, Paolo Degan, Anna Minoprio, Vito G Dagostino, Yoshihiro Matsumoto, Alessandra M Albertini, Margherita BignamiAbstract:MUTYH-associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8-hydroxyguanine (8-oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in MUTYH−/− mouse defective cells. Several parameters, including accumulation of 8-oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell-based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild-type protein. Our cell-based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity. Hum Mutat 30:1–8, 2009. © 2009 Wiley-Liss, Inc.
Ulrich Hübscher - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Human MUTYH DNA Glycosylase by the E3 Ubiquitin Ligase Mule
The Journal of biological chemistry, 2014Co-Authors: Julia Dorn, Elena Ferrari, Ralph Imhof, Nathalie Ziegler, Ulrich HübscherAbstract:Oxidation of DNA is a frequent and constantly occurring event. One of the best characterized oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxo-G). It instructs most DNA polymerases to preferentially insert an adenine (A) opposite 8-oxo-G instead of the appropriate cytosine (C) thus showing miscoding potential. The MutY DNA glycosylase homologue (MUTYH) recognizes A:8-oxo-G mispairs and removes the mispaired A giving way to the canonical base excision repair that ultimately restores undamaged guanine (G). Here we characterize for the first time in detail a posttranslational modification of the human MUTYH DNA glycosylase. We show that MUTYH is ubiquitinated in vitro and in vivo by the E3 ligase Mule between amino acids 475 and 535. Mutation of five lysine residues in this region significantly stabilizes MUTYH, suggesting that these are the target sites for ubiquitination. The endogenous MUTYH protein levels depend on the amount of expressed Mule. Furthermore, MUTYH and Mule physically interact. We found that a ubiquitination-deficient MUTYH mutant shows enhanced binding to chromatin. The mutation frequency of the ovarian cancer cell line A2780, analyzed at the HPRT locus can be increased upon oxidative stress and depends on the MUTYH levels that are regulated by Mule. This reflects the importance of tightly regulated MUTYH levels in the cell. In summary our data show that ubiquitination is an important regulatory mechanism for the essential MUTYH DNA glycosylase in human cells.
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MUTYH DNA glycosylase: the rationale for removing undamaged bases from the DNA.
Frontiers in genetics, 2013Co-Authors: Enni Markkanen, Julia Dorn, Ulrich HübscherAbstract:Maintenance of genetic stability is crucial for all organisms in order to avoid the onset of deleterious diseases such as cancer. One of the many proveniences of DNA base damage in mammalian cells is oxidative stress, arising from a variety of endogenous and exogenous sources, generating highly mutagenic oxidative DNA lesions. One of the best characterized oxidative DNA lesion is 7,8-dihydro-8-oxoguanine (8-oxo-G), which can give rise to base substitution mutations (also known as point mutations). This mutagenicity is due to the miscoding potential of 8-oxo-G that instructs most DNA polymerases (pols) to preferentially insert an Adenine (A) opposite 8-oxo-G instead of the appropriate Cytosine (C). If left unrepaired, such A:8-oxo-G mispairs can give rise to CG->AT transversion mutations. A:8-oxo-G mispairs are proficiently recognized by the MutY glycosylase homologue (MUTYH). MUTYH can remove the mispaired A from an A:8-oxo-G, giving way to the canonical base excision repair (BER) that ultimately restores undamaged Guanine (G). The importance of this MUTYH-initiated pathway is illustrated by the fact that biallelic mutations in the MUTYH gene are associated with a hereditary colorectal cancer syndrome termed MUTYH-associated polyposis (MAP). In this review, we will focus on MUTYH, from its discovery to the most recent data regarding its cellular roles and interaction partners. We discuss the involvement of the MUTYH protein in the A:8-oxo-G BER pathway acting together with pol , the pol that can faithfully incorporate C opposite 8-oxo-G and thus bypass this lesion in a correct manner. We also outline the current knowledge about the regulation of MUTYH itself and the A:8-oxo-G repair pathway by posttranslational modifications (PTM). Finally, to achieve a clearer overview of the literature, we will briefly touch on the rather confusing MUTYH nomenclature. In short, MUTYH is a unique DNA glycosylase that catalyzes the excision of an undamaged base from DNA.
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regulation of oxidative dna damage repair by dna polymerase λ and MUTYH by cross talk of phosphorylation and ubiquitination
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Enni Markkanen, Elena Ferrari, Barbara Van Loon, Jason L Parsons, Grigory L Dianov, Ulrich HübscherAbstract:It is of pivotal importance for genome stability that repair DNA polymerases (Pols), such as Pols λ and β, which all exhibit considerably reduced fidelity when replicating undamaged DNA, are tightly regulated, because their misregulation could lead to mutagenesis. Recently, we found that the correct repair of the abundant and highly miscoding oxidative DNA lesion 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-G) is performed by an accurate repair pathway that is coordinated by the MutY glycosylase homologue (MUTYH) and Pol λ in vitro and in vivo. Pol λ is phosphorylated by Cdk2/cyclinA in late S and G2 phases of the cell cycle, promoting Pol λ stability by preventing it from being targeted for proteasomal degradation by ubiquitination. However, it has remained a mystery how the levels of Pol λ are controlled, how phosphorylation promotes its stability, and how the engagement of Pol λ in active repair complexes is coordinated. Here, we show that the E3 ligase Mule mediates the degradation of Pol λ and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. Furthermore, we demonstrate that phosphorylation of Pol λ by Cdk2/cyclinA counteracts its Mule-mediated degradation by promoting recruitment of Pol λ to chromatin into active 8-oxo-G repair complexes through an increase in Pol λ's affinity to chromatin-bound MUTYH. Finally, MUTYH appears to promote the stability of Pol λ by binding it to chromatin. In contrast, Pol λ not engaged in active repair on chromatin is subject for proteasomal degradation.
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an 8 oxo guanine repair pathway coordinated by MUTYH glycosylase and dna polymerase λ
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Barbara Van Loon, Ulrich HübscherAbstract:Reactive oxygen species (ROS) interact with DNA, frequently generating highly mutagenic 7,8-dihydro-8-oxoguanine (8-oxo-G) lesions. Replicative DNA polymerases (pols) often misincorporate adenine opposite 8-oxo-G. The subsequent repair mechanism allowing the removal of adenine and formation of C:8-oxo-G base pair is essential to prevent C:G to A:T transversion mutations. Here, we show by immunofluorescence experiments, in cells exposed to ROS, the involvement of MutY glycosylase homologue (MUTYH) and DNA pol λ in the repair of A:8-oxo-G mispairs. We observe specific recruitment of MUTYH, DNA pol λ, proliferating cell nuclear antigen (PCNA), flap endonuclease 1 (FEN1) and DNA ligases I and III from human cell extracts to A:8-oxo-G DNA, but not to undamaged DNA. Using purified human proteins and a DNA template, we reconstitute the full pathway for the faithful repair of A:8-oxo-G mispairs involving MUTYH, DNA pol λ, FEN1, and DNA ligase I. These results reveal a cellular response pathway to ROS, important to sustain genomic stability and modulate carcinogenesis.
