RESUMEN

Alkyltransferase-like (ATL) proteins in Schizosaccharomyces pombe (Atl1) and Thermus thermophilus (TTHA1564) protect against the adverse effects of DNA alkylation damage by flagging O(6)-alkylguanine lesions for nucleotide excision repair (NER). We show that both ATL proteins bind with high affinity to oligodeoxyribonucleotides containing O(6)-alkylguanines differing in size, polarity, and charge of the alkyl group. However, Atl1 shows a greater ability than TTHA1564 to distinguish between O(6)-alkylguanine and guanine and in an unprecedented mechanism uses Arg69 to probe the electrostatic potential surface of O(6)-alkylguanine, as determined using molecular mechanics calculations. An unexpected consequence of this feature is the recognition of 2,6-diaminopurine and 2-aminopurine, as confirmed in crystal structures of respective Atl1-DNA complexes. O(6)-Alkylguanine and guanine discrimination is diminished for Atl1 R69A and R69F mutants, and S. pombe R69A and R69F mutants are more sensitive toward alkylating agent toxicity, revealing the key role of Arg69 in identifying O(6)-alkylguanines critical for NER recognition.

Asunto(s)

Transferasas Alquil y Aril/química , Reparación del ADN/fisiología , Guanina/química , Oligodesoxirribonucleótidos/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/enzimología , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Alquilación , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Guanina/metabolismo , Mutación Missense , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Thermus thermophilus/enzimología

RESUMEN

Nucleotide excision repair (NER) has long been known to remove DNA lesions induced by chemical carcinogens, and the molecular mechanism has been partially elucidated. Here we demonstrate that in Schizosaccharomyces pombe a DNA recognition protein, alkyltransferase-like 1 (Atl1), can play a pivotal role in selecting a specific NER pathway, depending on the nature of the DNA modification. The relative ease of dissociation of Atl1 from DNA containing small O(6)-alkylguanines allows accurate completion of global genome repair (GGR), whereas strong Atl1 binding to bulky O(6)-alkylguanines blocks GGR, stalls the transcription machinery, and diverts the damage to transcription-coupled repair. Our findings redraw the initial stages of the NER process in those organisms that express an alkyltransferase-like gene and raise the question of whether or not O(6)-alkylguanine lesions that are poor substrates for the alkyltransferase proteins in higher eukaryotes might, by analogy, signal such lesions for repair by NER.

Asunto(s)

Transferasas Alquil y Aril/metabolismo , Reparación del ADN , Guanina/análogos & derivados , Proteínas de Schizosaccharomyces pombe/metabolismo , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Western Blotting , Cristalografía por Rayos X , Daño del ADN , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/metabolismo , Citometría de Flujo , Fase G1/efectos de los fármacos , Genoma Fúngico/genética , Guanina/química , Guanina/metabolismo , Metilnitronitrosoguanidina/toxicidad , Modelos Moleculares , Mutación , Compuestos de Nitrosourea/toxicidad , Conformación de Ácido Nucleico , Unión Proteica , Estructura Terciaria de Proteína , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Transcripción Genética/genética

Asunto(s)

ADN/metabolismo , Conformación Proteica , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Biológicos , Unión Proteica

RESUMEN

Alkyltransferase-like proteins (ATLs) play a role in the protection of cells from the biological effects of DNA alkylation damage. Although ATLs share functional motifs with the DNA repair protein and cancer chemotherapy target O6-alkylguanine-DNA alkyltransferase, they lack the reactive cysteine residue required for alkyltransferase activity, so its mechanism for cell protection was previously unknown. Here we review recent advances in unraveling the enigmatic cellular protection provided by ATLs against the deleterious effects of DNA alkylation damage. We discuss exciting new evidence that ATLs aid in the repair of DNA O6-alkylguanine lesions through a novel repair cross-talk between DNA-alkylation base damage responses and the DNA nucleotide excision repair pathway.

Asunto(s)

Reparación del ADN , ADN/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Secuencia de Aminoácidos , Animales , ADN/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , O(6)-Metilguanina-ADN Metiltransferasa/química , Alineación de Secuencia , Transducción de Señal

RESUMEN

Alkyltransferase-like proteins (ATLs) are a novel class of DNA repair proteins related to O(6)-alkylguanine-DNA alkyltransferases (AGTs) that tightly bind alkylated DNA and shunt the damaged DNA into the nucleotide excision repair pathway. Here, we present the first structure of a bacterial ATL, from Vibrio parahaemolyticus (vpAtl). We demonstrate that vpAtl adopts an AGT-like fold and that the protein is capable of tightly binding to O(6)-methylguanine-containing DNA and disrupting its repair by human AGT, a hallmark of ATLs. Mutation of highly conserved residues Tyr(23) and Arg(37) demonstrate their critical roles in a conserved mechanism of ATL binding to alkylated DNA. NMR relaxation data reveal a role for conformational plasticity in the guanine-lesion recognition cavity. Our results provide further evidence for the conserved role of ATLs in this primordial mechanism of DNA repair.

Asunto(s)

Transferasas Alquil y Aril/química , Reparación del ADN/fisiología , ADN/química , Guanina/análogos & derivados , Pliegue de Proteína , Vibrio parahaemolyticus/enzimología , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Sustitución de Aminoácidos , ADN/genética , ADN/metabolismo , Guanina/química , Guanina/metabolismo , Humanos , Mutación Missense , Vibrio parahaemolyticus/genética

RESUMEN

O(6)-Alkylguanine-DNA alkyltransferase (AGT) plays a major role in repair of the cytotoxic and mutagenic lesion O(6)-methylguanine (m(6)G) in DNA. Unlike the Escherichia coli alkyltransferase Ogt that also repairs O(4)-methylthymine (m(4)T) efficiently, the human AGT (hAGT) acts poorly on m(4)T. Here we made several hAGT mutants in which residues near the cysteine acceptor site were replaced by corresponding residues from Ogt to investigate the basis for the inefficiency of hAGT in repair of m(4)T. Construct hAGT-03 (where hAGT sequence -V(149)CSSGAVGN(157)- was replaced with the corresponding Ogt -I(143)GRNGTMTG(151)-) exhibited enhanced m(4)T repair activity in vitro compared with hAGT. Three AGT proteins (hAGT, hAGT-03, and Ogt) exhibited similar protection from killing by N-methyl-N'-nitro-N-nitrosoguanidine and caused a reduction in m(6)G-induced G:C to A:T mutations in both nucleotide excision repair (NER)-proficient and -deficient Escherichia coli strains that lack endogenous AGTs. hAGT-03 resembled Ogt in totally reducing the m(4)T-induced T:A to C:G mutations in NER-proficient and -deficient strains. Surprisingly, wild type hAGT expression caused a significant but incomplete decrease in NER-deficient strains but a slight increase in T:A to C:G mutation frequency in NER-proficient strains. The T:A to C:G mutations due to O(4)-alkylthymine formed by ethylating and propylating agents were also efficiently reduced by either hAGT-03 or Ogt, whereas hAGT had little effect irrespective of NER status. These results show that specific alterations in the hAGT active site facilitate efficient recognition and repair of O(4)-alkylthymines and reveal damage-dependent interactions of base and nucleotide excision repair.

Asunto(s)

Aductos de ADN/metabolismo , Reparación del ADN/fisiología , O(6)-Metilguanina-ADN Metiltransferasa/genética , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Timina/metabolismo , Alquilación/fisiología , Dominio Catalítico , Daño del ADN/fisiología , Escherichia coli/genética , Etano/metabolismo , Humanos , Metano/metabolismo , Mutagénesis/fisiología , O(6)-Metilguanina-ADN Metiltransferasa/química , Propano/metabolismo , Estructura Terciaria de Proteína

RESUMEN

Alkyltransferase-like proteins (ATLs) share functional motifs with the cancer chemotherapy target O(6)-alkylguanine-DNA alkyltransferase (AGT) and paradoxically protect cells from the biological effects of DNA alkylation damage, despite lacking the reactive cysteine and alkyltransferase activity of AGT. Here we determine Schizosaccharomyces pombe ATL structures without and with damaged DNA containing the endogenous lesion O(6)-methylguanine or cigarette-smoke-derived O(6)-4-(3-pyridyl)-4-oxobutylguanine. These results reveal non-enzymatic DNA nucleotide flipping plus increased DNA distortion and binding pocket size compared to AGT. Our analysis of lesion-binding site conservation identifies new ATLs in sea anemone and ancestral archaea, indicating that ATL interactions are ancestral to present-day repair pathways in all domains of life. Genetic connections to mammalian XPG (also known as ERCC5) and ERCC1 in S. pombe homologues Rad13 and Swi10 and biochemical interactions with Escherichia coli UvrA and UvrC combined with structural results reveal that ATLs sculpt alkylated DNA to create a genetic and structural intersection of base damage processing with nucleotide excision repair.

Asunto(s)

Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Daño del ADN , Reparación del ADN , Alquilación , Sitios de Unión , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Guanina/análogos & derivados , Guanina/química , Guanina/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica

RESUMEN

O (6)-Alkylguanine-DNA alkyltransferase (AGT) plays an important role by protecting cells from alkylating agents. This reduces the frequency of carcinogenesis and mutagenesis initiated by such agents, but AGT also provides a major resistance mechanism to some chemotherapeutic drugs. To improve our understanding of the AGT-mediated repair reaction and our understanding of the spectrum of repairable damage, we have studied the ability of AGT to repair interstrand cross-link DNA damage where the two DNA strands are joined via the guanine- O (6) in each strand. An oligodeoxyribonucleotide containing a heptane cross-link was repaired with initial formation of an AGT-oligo complex and further reaction of a second AGT molecule yielding a hAGT dimer and free oligo. However, an oligodeoxyribonucleotide with a butane cross-link was a very poor substrate for AGT-mediated repair, and only the first reaction that forms an AGT-oligo complex could be detected. Models of the reaction of these substrates in the AGT active site show that the DNA duplex is forced apart locally to repair the first guanine. This reaction is greatly hindered with the butane cross-link, which is mostly buried in the active site pocket and limited in conformational flexibility. This limitation also prevents the adoption of a conformation for the second reaction to repair the AGT-oligo complex. These results are consistent with the postulated mechanism of AGT repair that involves DNA binding and flipping of the substrate nucleotide and indicate that hAGT can repair some types of interstrand cross-link damage.

Asunto(s)

Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Proteínas Supresoras de Tumor/metabolismo , Western Blotting , Cromatografía Líquida de Alta Presión , Daño del ADN , Metilasas de Modificación del ADN/química , Enzimas Reparadoras del ADN/química , Electroforesis en Gel de Poliacrilamida , Humanos , Modelos Moleculares , Proteínas Supresoras de Tumor/química

RESUMEN

O(6)-Alkylguanine-DNA alkyltransferase (AGT) is a crucial target both for the prevention of cancer and for chemotherapy, since it repairs mutagenic lesions in DNA, and it limits the effectiveness of alkylating chemotherapies. AGT catalyzes the unique, single-step, direct damage reversal repair of O(6)-alkylguanines by selectively transferring the O(6)-alkyl adduct to an internal cysteine residue. Recent crystal structures of human AGT alone and in complex with substrate DNA reveal a two-domain alpha/beta fold and a bound zinc ion. AGT uses its helix-turn-helix motif to bind substrate DNA via the minor groove. The alkylated guanine is then flipped out from the base stack into the AGT active site for repair by covalent transfer of the alkyl adduct to Cys145. An asparagine hinge (Asn137) couples the helix-turn-helix DNA binding and active site motifs. An arginine finger (Arg128) stabilizes the extrahelical DNA conformation. With this newly improved structural understanding of AGT and its interactions with biologically relevant substrates, we can now begin to unravel the role it plays in preserving genetic integrity and discover how it promotes resistance to anticancer therapies.

Asunto(s)

ADN/metabolismo , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Cristalografía por Rayos X , ADN/química , Metilasas de Modificación del ADN/química , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Reparación del ADN , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Resistencia a Antineoplásicos , Secuencias Hélice-Giro-Hélice , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Nucleótidos/química , O(6)-Metilguanina-ADN Metiltransferasa/química , O(6)-Metilguanina-ADN Metiltransferasa/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

The mature self-synthesizing p-hydroxybenzylideneimidazolinone-like fluorophores of Discosoma red fluorescent protein (DsRed) and Aequorea victoria green fluorescent protein (GFP) are extensively studied as powerful biological markers. Yet, the spontaneous formation of these fluorophores by cyclization, oxidation, and dehydration reactions of tripeptides within their protein environment remains incompletely understood. The mature DsRed fluorophore (Gln 66, Tyr 67, and Gly 68) differs from the GFP fluorophore by an acylimine that results in Gln 66 Calpha planar geometry and by a Phe 65-Gln 66 cis peptide bond. DsRed green-to-red maturation includes a green-fluorescing immature chromophore and requires a chromophore peptide bond trans-cis isomerization that is slow and incomplete. To clarify the unique structural chemistry for the individual immature "green" and mature "red" chromophores of DsRed, we report here the determination and analysis of crystal structures for the wild-type protein (1.4 A resolution), the entirely green DsRed K70M mutant protein (1.9 A resolution), and the DsRed designed mutant Q66M (1.9 A resolution), which shows increased red chromophore relative to the wild-type DsRed. Whereas the mature, red-fluorescing chromophore has the expected cis peptide bond and a sp(2)-hybridized Gln 66 Calpha with planar geometry, the crystal structure of the immature green-fluorescing chromophore of DsRed, presented here for the first time, reveals a trans peptide bond and a sp(3)-hybridized Gln 66 Calpha with tetrahedral geometry. These results characterize a GFP-like immature green DsRed chromophore structure, reveal distinct mature and immature chromophore environments, and furthermore provide evidence for the coupling of acylimine formation with trans-cis isomerization.

Asunto(s)

Antozoos , Proteínas Luminiscentes/química , Péptidos/química , Acilación , Animales , Antozoos/química , Antozoos/genética , Cristalización , Cristalografía por Rayos X , Proteínas Fluorescentes Verdes/química , Hidrozoos , Iminas/química , Isomerismo , Proteínas Luminiscentes/genética , Mutagénesis Sitio-Dirigida , Péptidos/genética , Proteína Fluorescente Roja