RESUMEN
Inherited defects of base excision repair have not been associated with any human genetic disorder, although mutations of the genes mutM and mutY, which function in Escherichia coli base excision repair, lead to increased transversions of G:C to T:A. We have studied family N, which is affected with multiple colorectal adenomas and carcinoma but lacks an inherited mutation of the adenomatous polyposis coli gene (APC) that is associated with familial adenomatous polyposis. Here we show that 11 tumors from 3 affected siblings contain 18 somatic inactivating mutations of APC and that 15 of these mutations are G:C-->A transversions--a significantly greater proportion than is found in sporadic tumors or in tumors associated with familial adenomatous polyposis. Analysis of the human homolog of mutY, MYH, showed that the siblings were compound heterozygotes for the nonconservative missense variants Tyr165Cys and Gly382Asp. These mutations affect residues that are conserved in mutY of E. coli (Tyr82 and Gly253). Tyrosine 82 is located in the pseudo-helix-hairpin-helix (HhH) motif and is predicted to function in mismatch specificity. Assays of adenine glycosylase activity of the Tyr82Cys and Gly253Asp mutant proteins with 8-oxoG:A and G:A substrates show that their activity is reduced significantly. Our findings link the inherited variants in MYH to the pattern of somatic APC mutation in family N and implicate defective base excision repair in predisposition to tumors in humans.
Asunto(s)
Neoplasias Colorrectales/genética , ADN Glicosilasas , N-Glicosil Hidrolasas/genética , Mutación Puntual , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Neoplasias Colorrectales/enzimología , Secuencia Conservada , Reparación del ADN/genética , ADN de Neoplasias/genética , Evolución Molecular , Femenino , Genes APC , Variación Genética , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The bacterial second messenger c-di-GMP is used in many species to control essential processes that allow the organism to adapt to its environment. The c-di-GMP riboswitch (GEMM) is an important downstream target in this signaling pathway and alters gene expression in response to changing concentrations of c-di-GMP. The riboswitch selectively recognizes its second messenger ligand primarily through contacts with two critical nucleotides. However, these two nucleotides are not the most highly conserved residues within the riboswitch sequence. Instead, nucleotides that stack with c-di-GMP and that form tertiary RNA contacts are the most invariant. Biochemical and structural evidence reveals that the most common natural variants are able to make alternative pairing interactions with both guanine bases of the ligand. Additionally, a high-resolution (2.3 A) crystal structure of the native complex reveals that a single metal coordinates the c-di-GMP backbone. Evidence is also provided that after transcription of the first nucleotide on the 3'-side of the P1 helix, which is predicted to be the molecular switch, the aptamer is functional for ligand binding. Although large energetic effects occur when several residues in the RNA are altered, mutations at the most conserved positions, rather than at positions that base pair with c-di-GMP, have the most detrimental effects on binding. Many mutants retain sufficient c-di-GMP affinity for the RNA to remain biologically relevant, which suggests that this motif is quite resilient to mutation.
Asunto(s)
Guanosina Monofosfato/metabolismo , Emparejamiento Base , Secuencia de Bases , GMP Cíclico/análogos & derivados , Guanina/química , Guanina/metabolismo , Ligandos , Mutación , Estructura Terciaria de Proteína , Sistemas de Mensajero Secundario/fisiologíaRESUMEN
Escherichia coli MutY has an important role in preventing mutations associated with the oxidative lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) in DNA by excising adenines from OG.A mismatches as the first step of base excision repair. To determine the importance of specific steps in the base pair recognition and base removal process of MutY, we have evaluated the effects of modifications of the OG.A substrate on the kinetics of base removal, mismatch affinity and repair to G-C in an E. coli-based assay. Notably, adenine modification was tolerated in the cellular assay, whereas modification of OG resulted in minimal cellular repair. High affinity for the mismatch and efficient base removal required the presence of OG. Taken together, these results suggest that the presence of OG is a critical feature that is necessary for MutY to locate OG.A mismatches and select the appropriate adenines for excision to initiate repair in vivo before replication.
Asunto(s)
Adenina/metabolismo , Daño del ADN , ADN Glicosilasas/fisiología , Reparación de la Incompatibilidad de ADN , Desoxiguanosina/análogos & derivados , Escherichia coli , Guanina/análogos & derivados , 8-Hidroxi-2'-Desoxicoguanosina , Emparejamiento Base , Secuencia de Bases , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Desoxiguanosina/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Guanina/metabolismo , Cinética , Datos de Secuencia Molecular , Oligonucleótidos/química , Oligonucleótidos/genética , Plásmidos , Especificidad por SustratoRESUMEN
The oxidized guanine lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) is highly mutagenic, resulting in G:C to T:A transversion mutations in the absence of repair. The Escherichia coli adenine glycosylase MutY and its human homolog (hMYH) play an important role in the prevention of mutations associated with OG by removing misincorporated adenine residues from OG:A mismatches. Previously, biallelic mutations of hMYH have been identified in a British family (Family N) with symptoms characteristic of familial adenomatous polyposis (FAP), which is typically associated with mutations in the adenomatous polyposis coli (APC) gene. Afflicted members of this family were compound heterozygotes for two mutations in hMYH, Y165C and G382D. These positions are highly conserved in MutY across phylogeny. The current work reveals a reduced ability of the hMYH variants compared to wild-type (WT) hMYH to complement the activity of E.coli MutY in mutY((-)) E.coli. In vitro analysis of the corresponding mutations in E.coli MutY revealed a reduction in the adenine glycosylase activity of the enzymes. In addition, evaluation of substrate affinity using a substrate analog, 2'-deoxy-2'-fluoroadenosine (FA) revealed that both mutations severely diminish the ability to recognize FA, and discriminate between OG and G. Importantly, adenine removal with both the mutant and WT E.coli enzymes was observed to be less efficient from a mismatch in the sequence context observed to be predominantly mutated in tumors of Family N. Interestingly, the magnitude of the reduced activity of the E.coli mutant enzymes relative to the WT enzyme was magnified in the "hotspot" sequence context. If the corresponding mutations in hMYH cause similar sensitivity to sequence context, this effect may contribute to the specific targeting of the APC gene. The lack of complementation of the hMYH variants for MutY, and the reduced activity of the Y82C and G253D E.coli enzymes, provide additional circumstantial evidence that the somatic mutations in APC, and the occurrence of FAP in Family N, are due to a reduced ability of the Y165C and G382D hMYH enzymes to recognize and repair OG:A mismatches.
Asunto(s)
Poliposis Adenomatosa del Colon/enzimología , Neoplasias Colorrectales/enzimología , ADN Glicosilasas , Desoxiguanosina/análogos & derivados , Escherichia coli/enzimología , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Adenina , Disparidad de Par Base , Sitios de Unión , Cartilla de ADN/química , Reparación del ADN , Desoxiadenosinas/farmacología , Desoxiguanosina/farmacología , Genes APC/fisiología , Prueba de Complementación Genética , Variación Genética , Guanina , Heterocigoto , Humanos , Técnicas In Vitro , Cinética , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la PolimerasaRESUMEN
MUTYH-associated polyposis (MAP) is the only inherited colorectal cancer syndrome that is associated with inherited biallelic mutations in a base excision repair gene. The MUTYH glycosylase plays an important role in preventing mutations associated with 8-oxoguanine (OG) by removing adenine residues that have been misincorporated opposite OG. MAP-associated mutations are present throughout MUTYH, with a large number coding for missense variations. To date the available information on the functional properties of MUTYH variants is conflicting. In this study, a kinetic analysis of the adenine glycosylase activity of MUTYH and several variants was undertaken using a correction for active fraction to control for differences due to overexpression and purification. Using these methods, the rate constants for steps involved in the adenine removal process were determined for the MAP variants Y165C, G382D, P391L and Q324R MUTYH. Under single-turnover conditions, the rate of adenine removal for these four variants was found to be 30-40% of WT MUTYH. In addition, the ability of MUTYH and the variants to suppress mutations and complement for the absence of MutY in Escherichia coli was assessed using rifampicin resistance assays. The presence of WT and Q324R MUTYH resulted in complete suppression of the mutation frequency, while G382D MUTYH showed reduced ability to suppress the mutation frequency. In contrast, the mutation frequency observed upon expression of P391L and Y165C MUTYH were similar to the controls, suggesting no activity toward preventing DNA mutations. Notably, though all variations studied herein resulted in similar reductions in adenine glycosylase activity, the effects in the bacterial complementation are quite different. This suggests that the consequences of a specific amino acid variation on overall repair in a cellular context may be magnified.
Asunto(s)
Adenina/metabolismo , Neoplasias del Colon/enzimología , ADN Glicosilasas/metabolismo , Escherichia coli/enzimología , Mutación , Dominio Catalítico , Neoplasias del Colon/genética , Cristalografía por Rayos X , ADN Glicosilasas/química , ADN Glicosilasas/genética , Estabilidad de Enzimas , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
The oxidation product of 2'-deoxyguanosine, 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG), produces G:C to T:A transversion mutations. The Escherichia coli base excision repair glycosylase MutY plays an important role in preventing OG-associated mutations by removing adenines misincorporated opposite OG lesions during DNA replication. Recently, biallelic mutations in the human MutY homologue (hMYH) have been correlated with the development of colorectal cancer. The two most common mutations correspond to two single amino acid substitutions in the hMYH protein: Y165C and G382D [Al-Tassan, N., et al. (2002) Nat. Genet. 30, 227-232]. Previously, our laboratory analyzed the adenine glycosylase activity of the homologous variant E. coli MutY enzymes, Y82C and G253D [Chmiel, N. H., et al. (2003) J. Mol. Biol. 327, 431-443]. This work demonstrated that both variants have a reduced adenine glycosylase activity and affinity for substrate analogues compared to wild-type MutY. Recent structural work on Bacillus stearothermophilus MutY bound to an OG:A mismatch-containing duplex indicates that both residues aid in recognition of OG [Fromme, J. C., et al. (2004) Nature 427, 652-656]. To determine the extent with which Tyr 82 and Gly 253 contribute to catalysis of adenine removal by E. coli MutY, we made a series of additional modifications in these residues, namely, Y82F, Y82L, and G253A. When the substrate analogue 2'-deoxy-2'-fluoroadenosine (FA) in duplex paired with G or OG is used, both Y82F and G253A showed reduced binding affinity, and G253A was unable to discriminate between OG and G when paired with FA. Additionally, compromised glycosylase activity of Y82F, Y82C, and G253A MutY was observed using the nonoptimal G:A substrate, or at low reaction temperatures. Interestingly, adenine removal from an OG:A-containing DNA substrate by Y82C MutY was also shown to be extremely sensitive to the NaCl concentration. The most surprising result was the remarkably similar activity of Y82L MutY to the WT enzyme under all conditions examined, indicating that a leucine residue may effectively replace tyrosine for intercalation at the OG:A mismatch. The results contained herein provide further insight regarding the intricate roles of Tyr 82 and Gly 253 in the OG recognition and adenine excision functions of MutY.
Asunto(s)
ADN Glicosilasas/metabolismo , Escherichia coli/enzimología , Glicina/química , N-Glicosil Hidrolasas/metabolismo , Tirosina/química , Sustitución de Aminoácidos , Disparidad de Par Base , Sitios de Unión , ADN/genética , ADN Glicosilasas/genética , Desoxiadenosinas/química , Humanos , Estructura Molecular , Mutación , N-Glicosil Hidrolasas/genéticaRESUMEN
DNA charge transport (CT) chemistry provides a route to carry out oxidative DNA damage from a distance in a reaction that is sensitive to DNA mismatches and lesions. Here, DNA-mediated CT also leads to oxidation of a DNA-bound base excision repair enzyme, MutY. DNA-bound Ru(III), generated through a flash/quench technique, is found to promote oxidation of the [4Fe-4S](2+) cluster of MutY to [4Fe-4S](3+) and its decomposition product [3Fe-4S](1+). Flash/quench experiments monitored by EPR spectroscopy reveal spectra with g = 2.08, 2.06, and 2.02, characteristic of the oxidized clusters. Transient absorption spectra of poly(dGC) and [Ru(phen)(2)dppz](3+) (dppz = dipyridophenazine), generated in situ, show an absorption characteristic of the guanine radical that is depleted in the presence of MutY with formation instead of a long-lived species with an absorption at 405 nm; we attribute this absorption also to formation of the oxidized [4Fe-4S](3+) and [3Fe-4S](1+) clusters. In ruthenium-tethered DNA assemblies, oxidative damage to the 5'-G of a 5'-GG-3' doublet is generated from a distance but this irreversible damage is inhibited by MutY and instead EPR experiments reveal cluster oxidation. With ruthenium-tethered assemblies containing duplex versus single-stranded regions, MutY oxidation is found to be mediated by the DNA duplex, with guanine radical as an intermediate oxidant; guanine radical formation facilitates MutY oxidation. A model is proposed for the redox activation of DNA repair proteins through DNA CT, with guanine radicals, the first product under oxidative stress, in oxidizing the DNA-bound repair proteins, providing the signal to stimulate DNA repair.
Asunto(s)
ADN Glicosilasas/metabolismo , Reparación del ADN , ADN/metabolismo , Transporte de Electrón , Guanina/metabolismo , Daño del ADN , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática , Radicales Libres , Oxidación-ReducciónRESUMEN
MutY, like many DNA base excision repair enzymes, contains a [4Fe4S]2+ cluster of undetermined function. Electrochemical studies of MutY bound to a DNA-modified gold electrode demonstrate that the [4Fe4S] cluster of MutY can be accessed in a DNA-mediated redox reaction. Although not detectable without DNA, the redox potential of DNA-bound MutY is approximately 275 mV versus NHE, which is characteristic of HiPiP iron proteins. Binding to DNA is thus associated with a change in [4Fe4S]3+/2+ potential, activating the cluster toward oxidation. Given that DNA charge transport chemistry is exquisitely sensitive to perturbations in base pair structure, such as mismatches, we propose that this redox process of MutY bound to DNA exploits DNA charge transport and provides a DNA signaling mechanism to scan for mismatches and lesions in vivo.