RESUMEN
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 and C-terminal 8-oxoG recognition domains. In this work, we identified a candidate for the fourth Zn2+ coordinating ligand using a combination of bioinformatics and computational modeling. In addition, using in vitro enzyme activity assays, fluorescence polarization DNA binding assays, circular dichroism spectroscopy, and cell-based rifampicin resistance assays, the functional impact of reduced Zn2+ chelation was evaluated. Taken together, these results illustrate the critical role that the "Zn2+ linchpin motif" plays in MUTYH repair activity by providing for proper engagement of the functional domains on the 8-oxoG:A mismatch required for base excision catalysis. The functional importance of the Zn2+ linchpin also suggests that adjacent MAP variants or exposure to environmental chemicals may compromise Zn2+ coordination, and ability of MUTYH to prevent disease.
Asunto(s)
ADN Glicosilasas/metabolismo , Zinc/metabolismo , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Cisteína/química , ADN Glicosilasas/química , ADN Glicosilasas/genética , Geobacillus stearothermophilus/enzimología , Humanos , Ligandos , Ratones , Mutación , Unión Proteica , Alineación de SecuenciaRESUMEN
A growing number of iron-sulfur (Fe-S) cluster cofactors have been identified in DNA repair proteins. MutY and its homologs are base excision repair (BER) glycosylases that prevent mutations associated with the common oxidation product of guanine (G), 8-oxo-7,8-dihydroguanine (OG) by catalyzing adenine (A) base excision from inappropriately formed OG:A mispairs. The finding of an [4Fe-4S]2+ cluster cofactor in MutY, Endonuclease III, and structurally similar BER enzymes was surprising and initially thought to represent an example of a purely structural role for the cofactor. However, in the two decades subsequent to the initial discovery, purification and in vitro analysis of bacterial MutYs and mammalian homologs, such as human MUTYH and mouse Mutyh, have demonstrated that proper Fe-S cluster coordination is required for OG:A substrate recognition and adenine excision. In addition, the Fe-S cluster in MutY has been shown to be capable of redox chemistry in the presence of DNA. The work in our laboratory aimed at addressing the importance of the MutY Fe-S cluster has involved a battery of approaches, with the overarching hypothesis that understanding the role(s) of the Fe-S cluster is intimately associated with understanding the biological and chemical properties of MutY and its unique damaged DNA substrate as a whole. In this chapter, we focus on methods of enzyme expression and purification, detailed enzyme kinetics, and DNA affinity assays. The methods described herein have not only been leveraged to provide insight into the roles of the MutY Fe-S cluster but have also been provided crucial information needed to delineate the impact of inherited variants of the human homolog MUTYH associated with a colorectal cancer syndrome known as MUTYH-associated polyposis or MAP. Notably, many MAP-associated variants have been found adjacent to the Fe-S cluster further underscoring the intimate relationship between the cofactor, MUTYH-mediated DNA repair, and disease.
Asunto(s)
Clonación Molecular/métodos , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , ADN/metabolismo , Pruebas de Enzimas/métodos , Animales , ADN/química , Daño del ADN , ADN Glicosilasas/química , Reparación del ADN , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/enzimología , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Humanos , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Cinética , Ratones , Modelos MolecularesRESUMEN
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.
Asunto(s)
ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Reparación del ADN , Animales , Antibacterianos/farmacología , Clonación Molecular/métodos , Farmacorresistencia Bacteriana , Pruebas de Enzimas/métodos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Modelos Moleculares , Mutación , Tasa de Mutación , Rifampin/farmacologíaRESUMEN
S K-edge X-ray absorption spectroscopy (XAS) was used to study the [Fe4S4] clusters in the DNA repair glycosylases EndoIII and MutY to evaluate the effects of DNA binding and solvation on Fe-S bond covalencies (i.e., the amount of S 3p character mixed into the Fe 3d valence orbitals). Increased covalencies in both iron-thiolate and iron-sulfide bonds would stabilize the oxidized state of the [Fe4S4] clusters. The results are compared to those on previously studied [Fe4S4] model complexes, ferredoxin (Fd), and to new data on high-potential iron-sulfur protein (HiPIP). A limited decrease in covalency is observed upon removal of solvent water from EndoIII and MutY, opposite to the significant increase observed for Fd, where the [Fe4S4] cluster is solvent exposed. Importantly, in EndoIII and MutY, a large increase in covalency is observed upon DNA binding, which is due to the effect of its negative charge on the iron-sulfur bonds. In EndoIII, this change in covalency can be quantified and makes a significant contribution to the observed decrease in reduction potential found experimentally in DNA repair proteins, enabling their HiPIP-like redox behavior.
Asunto(s)
ADN Glicosilasas/metabolismo , ADN/metabolismo , Desoxirribonucleasa (Dímero de Pirimidina)/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Geobacillus stearothermophilus/enzimología , Bacterias/química , Bacterias/enzimología , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , ADN Glicosilasas/química , Desoxirribonucleasa (Dímero de Pirimidina)/química , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Modelos Moleculares , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Unión Proteica , Espectroscopía de Absorción de Rayos X/métodosRESUMEN
Escherichia coli endonuclease III (EndoIII) and MutY are DNA glycosylases that contain [4Fe4S] clusters and that serve to maintain the integrity of the genome after oxidative stress. Electrochemical studies on highly oriented pyrolytic graphite (HOPG) revealed that DNA binding by EndoIII leads to a large negative shift in the midpoint potential of the cluster, consistent with stabilization of the oxidized [4Fe4S]3+ form. However, the smooth, hydrophobic HOPG surface is nonideal for working with proteins in the absence of DNA. In this work, we use thin film voltammetry on a pyrolytic graphite edge electrode to overcome these limitations. Improved adsorption leads to substantial signals for both EndoIII and MutY in the absence of DNA, and a large negative potential shift is retained with DNA present. In contrast, the EndoIII mutants E200K, Y205H, and K208E, which provide electrostatic perturbations in the vicinity of the cluster, all show DNA-free potentials within error of wild type; similarly, the presence of negatively charged poly-l-glutamate does not lead to a significant potential shift. Overall, binding to the DNA polyanion is the dominant effect in tuning the redox potential of the [4Fe4S] cluster, helping to explain why all DNA-binding proteins with [4Fe4S] clusters studied to date have similar DNA-bound potentials.
Asunto(s)
Reparación del ADN , ADN , ADN Glicosilasas , Técnicas Electroquímicas , Hierro , Oxidación-Reducción , AzufreRESUMEN
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.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Neoplasias Colorrectales/genética , ADN Glicosilasas/metabolismo , Reparación del ADN , ADN/metabolismo , Guanina/análogos & derivados , Animales , Catálisis , ADN/química , ADN Glicosilasas/genética , Predisposición Genética a la Enfermedad , Guanina/química , Guanina/metabolismo , Humanos , Metales/química , Metales/metabolismo , Mutación/genética , Polimorfismo Genético , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The important industrial and environmental carcinogen 1,3-butadiene (BD) forms a range of adenine adducts in DNA, including N6-(2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (N6-HB-dA), 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6-HMHP-dA), and N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N6,N6-DHB-dA). If not removed prior to DNA replication, these lesions can contribute to A â T and A â G mutations commonly observed following exposure to BD and its metabolites. In this study, base excision repair of BD-induced 2'-deoxyadenosine (BD-dA) lesions was investigated. Synthetic DNA duplexes containing site-specific and stereospecific (S)-N6-HB-dA, (R,S)-1,N6-HMHP-dA, and (R,R)-N6,N6-DHB-dA adducts were prepared by a postoligomerization strategy. Incision assays with nuclear extracts from human fibrosarcoma (HT1080) cells have revealed that BD-dA adducts were recognized and cleaved by a BER mechanism, with the relative excision efficiency decreasing in the following order: (S)-N6-HB-dA > (R,R)-N6,N6-DHB-dA > (R,S)-1,N6-HMHP-dA. The extent of strand cleavage at the adduct site was decreased in the presence of BER inhibitor methoxyamine and by competitor duplexes containing known BER substrates. Similar strand cleavage assays conducted using several eukaryotic DNA glycosylases/lyases (AAG, Mutyh, hNEIL1, and hOGG1) have failed to observe correct incision products at the BD-dA lesion sites, suggesting that a different BER enzyme may be involved in the removal of BD-dA adducts in human cells.
Asunto(s)
Butadienos/química , Reparación del ADN , Desoxiadenosinas/química , Animales , Línea Celular , Línea Celular Tumoral , Cricetinae , Cricetulus , HumanosRESUMEN
Aberrant epigenetic methylation is linked to the onset and progression of cancer. In this issue of Chemistry & Biology, Furst and Barton (2015) describe a sensitive electrochemical assay that can detect hyperactive epigenetic methylation in tumor tissue.