RESUMEN
Activation-induced cytidine deaminase (AID) interacts with replication protein A (RPA), the major ssDNA-binding protein, to promote deamination of cytosine to uracil in transcribed immunoglobulin (Ig) genes. Uracil-DNA glycosylase (UNG) acts in concert with AID during Ig diversification. In addition, UNG preserves genome integrity by base-excision repair (BER) in the overall genome. How UNG is regulated to support both mutagenic processing and error-free repair remains unknown. UNG is expressed as two isoforms, UNG1 and UNG2, which both contain an RPA-binding helix that facilitates uracil excision from RPA-coated ssDNA. However, the impact of this interaction in antibody diversification and genome maintenance has not been investigated. Here, we generated B-cell clones with targeted mutations in the UNG RPA-binding motif, and analysed class switch recombination (CSR), mutation frequency (5' Ig Sµ), and genomic uracil in clones representing seven Ung genotypes. We show that the UNG:RPA interaction plays a crucial role in both CSR and repair of AID-induced uracil at the Ig loci. By contrast, the interaction had no significant impact on total genomic uracil levels. Thus, RPA coordinates UNG during CSR and pre-replicative repair of mutagenic uracil in ssDNA but is not essential in post-replicative and canonical BER of uracil in dsDNA.
Asunto(s)
Proteína de Replicación A , Uracil-ADN Glicosidasa , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Reparación del ADN/genética , ADN de Cadena Simple/genética , Cambio de Clase de Inmunoglobulina/genética , Isotipos de Inmunoglobulinas/genética , Inmunoglobulinas/genética , Mutágenos , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Uracilo/metabolismo , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/metabolismo , Humanos , Animales , RatonesRESUMEN
Uracil occurs at replication forks via misincorporation of deoxyuridine monophosphate (dUMP) or via deamination of existing cytosines, which occurs 2-3 orders of magnitude faster in ssDNA than in dsDNA and is 100% miscoding. Tethering of UNG2 to proliferating cell nuclear antigen (PCNA) allows rapid post-replicative removal of misincorporated uracil, but potential 'pre-replicative' removal of deaminated cytosines in ssDNA has been questioned since this could mediate mutagenic translesion synthesis and induction of double-strand breaks. Here, we demonstrate that uracil-DNA glycosylase (UNG), but not SMUG1 efficiently excises uracil from replication protein A (RPA)-coated ssDNA and that this depends on functional interaction between the flexible winged-helix (WH) domain of RPA2 and the N-terminal RPA-binding helix in UNG. This functional interaction is promoted by mono-ubiquitination and diminished by cell-cycle regulated phosphorylations on UNG. Six other human proteins bind the RPA2-WH domain, all of which are involved in DNA repair and replication fork remodelling. Based on this and the recent discovery of the AP site crosslinking protein HMCES, we propose an integrated model in which templated repair of uracil and potentially other mutagenic base lesions in ssDNA at the replication fork, is orchestrated by RPA. The UNG:RPA2-WH interaction may also play a role in adaptive immunity by promoting efficient excision of AID-induced uracils in transcribed immunoglobulin loci.
Asunto(s)
ADN Glicosilasas/metabolismo , Replicación del ADN , ADN de Cadena Simple/metabolismo , Proteína de Replicación A/metabolismo , Uracilo/metabolismo , Sitios de Unión , Humanos , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
Human uracil N-glycosylase isoform 2-UNG2 consists of an N-terminal intrinsically disordered regulatory domain (UNG2 residues 1-92, 9.3 kDa) and a C-terminal structured catalytic domain (UNG2 residues 93-313, 25.1 kDa). Here, we report the backbone 1H, 13C, and 15N chemical shift assignment as well as secondary structure analysis of the N-and C-terminal domains of UNG2 representing the full-length UNG2 protein.
Asunto(s)
ADN Glicosilasas/química , Resonancia Magnética Nuclear Biomolecular , ADN Glicosilasas/metabolismo , Humanos , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
Considerable progress has been made in understanding the origins of genomic uracil and its role in genome stability and host defense; however, the main question concerning the basal level of uracil in DNA remains disputed. Results from assays designed to quantify genomic uracil vary by almost three orders of magnitude. To address the issues leading to this inconsistency, we explored possible shortcomings with existing methods and developed a sensitive LC/MS/MS-based method for the absolute quantification of genomic 2'-deoxyuridine (dUrd). To this end, DNA was enzymatically hydrolyzed to 2'-deoxyribonucleosides and dUrd was purified in a preparative HPLC step and analyzed by LC/MS/MS. The standard curve was linear over four orders of magnitude with a quantification limit of 5 fmol dUrd. Control samples demonstrated high inter-experimental accuracy (94.3%) and precision (CV 9.7%). An alternative method that employed UNG2 to excise uracil from DNA for LC/MS/MS analysis gave similar results, but the intra-assay variability was significantly greater. We quantified genomic dUrd in Ung(+/+) and Ung(-/-) mouse embryonic fibroblasts and human lymphoblastoid cell lines carrying UNG mutations. DNA-dUrd is 5-fold higher in Ung(-/-) than in Ung(+/+) fibroblasts and 11-fold higher in UNG2 dysfunctional than in UNG2 functional lymphoblastoid cells. We report approximately 400-600 dUrd per human or murine genome in repair-proficient cells, which is lower than results using other methods and suggests that genomic uracil levels may have previously been overestimated.
Asunto(s)
ADN/química , Nucleótidos de Uracilo/química , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , ADN/genética , ADN/aislamiento & purificación , Contaminación de ADN , Genoma Humano , Humanos , Hidrólisis , Límite de Detección , Ratones , Ratones Noqueados , Estándares de Referencia , Espectrometría de Masas en Tándem/normas , Nucleótidos de Uracilo/genética , Nucleótidos de Uracilo/aislamiento & purificación , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/metabolismoRESUMEN
Genome-wide gene expression analyses of the human somatic cell cycle have indicated that the set of cycling genes differ between primary and cancer cells. By identifying genes that have cell cycle dependent expression in HaCaT human keratinocytes and comparing these with previously identified cell cycle genes, we have identified three distinct groups of cell cycle genes. First, housekeeping genes enriched for known cell cycle functions; second, cell type-specific genes enriched for HaCaT-specific functions; and third, Polycomb-regulated genes. These Polycomb-regulated genes are specifically upregulated during DNA replication, and consistent with being epigenetically silenced in other cell cycle phases, these genes have lower expression than other cell cycle genes. We also find similar patterns in foreskin fibroblasts, indicating that replication-dependent expression of Polycomb-silenced genes is a prevalent but unrecognized regulatory mechanism.
Asunto(s)
Ciclo Celular/genética , Replicación del ADN , Proteínas del Grupo Polycomb/fisiología , Regulación hacia Arriba , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Islas de CpG , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Genes Esenciales , Histonas/fisiología , Humanos , Queratinocitos/metabolismo , Queratinocitos/fisiología , Análisis de los Mínimos Cuadrados , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética , TranscriptomaRESUMEN
Cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions induced in DNA and RNA in vitro and in pre-damaged DNA and RNA bacteriophages in vivo are repaired by the Escherichia coli (E. coli) protein AlkB and a human homolog, ALKBH3. However, it is not known whether endogenous RNA is repaired in vivo by repair proteins present at physiological concentrations. The concept of RNA repair as a biologically relevant process has therefore remained elusive. Here, we demonstrate AlkB-mediated repair of endogenous RNA in vivo by measuring differences in lesion-accumulation in two independent AlkB-proficient and deficient E. coli strains during exposure to methyl methanesulfonate (MMS). Repair was observed both in AlkB-overproducing strains and in the wild-type strains after AlkB induction. RNA repair appeared to be highest in RNA species below 200 nucleotides in size, mainly comprising tRNAs. Strikingly, at least 10-fold more lesions were repaired in RNA than in DNA. This may be a consequence of some 30-fold higher levels of aberrant methylation in RNA than in DNA after exposure to MMS. A high primary kinetic isotope effect (>10) was measured using a deuterated methylated RNA substrate, D3-1me(rA), demonstrating that it is the catalytic step, and not the search step that is rate-limiting. Our results demonstrate that RNA repair by AlkB takes place in endogenous RNA as part of an adaptive response in wild-type E. coli cells.
Asunto(s)
Proteínas de Escherichia coli/fisiología , Escherichia coli/enzimología , Oxigenasas de Función Mixta/fisiología , ARN Bacteriano/genética , Adaptación Fisiológica/genética , Alquilantes/farmacología , Reparación del ADN , ADN Bacteriano/genética , Inducción Enzimática , Escherichia coli/genética , Cinética , Metilmetanosulfonato/farmacología , Metilación , ARN Bacteriano/metabolismoRESUMEN
Human nuclear uracil-DNA glycosylase UNG2 is essential for post-replicative repair of uracil in DNA, and UNG2 protein and mRNA levels rapidly decline in G2/M phase. Previous work has demonstrated regulation of UNG2 at the transcriptional level, as well as by protein phosphorylation and ubiquitylation. UNG2 mRNA, encoded by the UNG gene, contains a long 3'untranslated region (3'UTR) of previously unknown function. Here, we demonstrate that several conserved regions in the 3'UTR are potential seed sites for microRNAs (miRNAs), such as miR-16, miR-34c, and miR-199a. Our results show that these miRNAs down-regulate UNG activity, UNG mRNA, and UNG protein levels. Down-regulation was dependent on the 3'UTR, indicating that the miRNAs directly target the conserved seed sites in the 3'UTR. These results add miRNAs as a new modality to UNG's increasing list of complex regulatory mechanisms.
Asunto(s)
Regulación hacia Abajo , MicroARNs/metabolismo , Uracil-ADN Glicosidasa/metabolismo , Regiones no Traducidas 3' , Células HeLa , Humanos , Uracil-ADN Glicosidasa/genéticaRESUMEN
BACKGROUND: Preeclampsia is a serious pregnancy complication, demonstrating a complex pattern of inheritance. The elucidation of genetic liability to preeclampsia remains a major challenge in obstetric medicine. We have adopted a positional cloning approach to identify maternal genetic components, with linkages previously demonstrated to chromosomes 2q, 5q and 13q in an Australian/New Zealand familial cohort. The current study aimed to identify potential functional and structural variants in the positional candidate gene TNFSF13B under the 13q linkage peak and assess their association status with maternal preeclampsia genetic susceptibility. METHODOLOGY/PRINCIPAL FINDINGS: The proximal promoter and coding regions of the positional candidate gene TNFSF13B residing within the 13q linkage region was sequenced using 48 proband or founder individuals from Australian/New Zealand families. Ten sequence variants (nine SNPs and one single base insertion) were identified and seven SNPs were successfully genotyped in the total Australian/New Zealand family cohort (74 families/480 individuals). Borderline association to preeclampsia (p = 0.0153) was observed for three rare SNPs (rs16972194, rs16972197 and rs56124946) in strong linkage disequilibrium with each other. Functional evaluation by electrophoretic mobility shift assays showed differential nuclear factor binding to the minor allele of the rs16972194 SNP, residing upstream of the translation start site, making this a putative functional variant. The observed genetic associations were not replicated in a Norwegian case/control cohort (The Nord-Trøndelag Health Study (HUNT2), 851 preeclamptic and 1,440 non-preeclamptic women). CONCLUSION/SIGNIFICANCE: TNFSF13B has previously been suggested to contribute to the normal immunological adaption crucial for a successful pregnancy. Our observations support TNFSF13B as a potential novel preeclampsia susceptibility gene. We discuss a possible role for TNFSF13B in preeclampsia pathogenesis, and propose the rs16972194 variant as a candidate for further functional evaluation.
Asunto(s)
Factor Activador de Células B/genética , Cromosomas Humanos Par 13/genética , Predisposición Genética a la Enfermedad , Variación Genética , Preeclampsia/genética , Australia , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Humanos , Noruega , Polimorfismo de Nucleótido Simple , Embarazo , Población Blanca/genéticaRESUMEN
Numerous proteins, many essential for the DNA replication machinery, interact with proliferating cell nuclear antigen (PCNA) through the PCNA-interacting peptide (PIP) sequence called the PIP box. We have previously shown that the oxidative demethylase human AlkB homologue 2 (hABH2) colocalizes with PCNA in replication foci. In this study, we show that hABH2 interacts with a posttranslationally modified PCNA via a novel PCNA-interacting motif, which we term AlkB homologue 2 PCNA-interacting motif (APIM). We identify APIM in >200 other proteins involved in DNA maintenance, transcription, and cell cycle regulation, and verify a functional APIM in five of these. Expression of an APIM peptide increases the cellular sensitivity to several cytostatic agents not accounted for by perturbing only the hABH2-PCNA interaction. Thus, APIM is likely to mediate PCNA binding in many proteins involved in DNA repair and cell cycle control during genotoxic stress.
Asunto(s)
Secuencias de Aminoácidos/genética , Enzimas Reparadoras del ADN/metabolismo , Dioxigenasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB , Animales , Reparación del ADN , Enzimas Reparadoras del ADN/genética , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Dioxigenasas/genética , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Antígeno Nuclear de Célula en Proliferación/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Factores de Transcripción TFII/genética , Factores de Transcripción TFII/metabolismoRESUMEN
Methylating agents are ubiquitous in the environment, and central in cancer therapy. The 1-methyladenine and 3-methylcytosine lesions in DNA/RNA contribute to the cytotoxicity of such agents. These lesions are directly reversed by ABH3 (hABH3) in humans and AlkB in Escherichia coli. Here, we report the structure of the hABH3 catalytic core in complex with iron and 2-oxoglutarate (2OG) at 1.5 A resolution and analyse key site-directed mutants. The hABH3 structure reveals the beta-strand jelly-roll fold that coordinates a catalytically active iron centre by a conserved His1-X-Asp/Glu-X(n)-His2 motif. This experimentally establishes hABH3 as a structural member of the Fe(II)/2OG-dependent dioxygenase superfamily, which couples substrate oxidation to conversion of 2OG into succinate and CO2. A positively charged DNA/RNA binding groove indicates a distinct nucleic acid binding conformation different from that predicted in the AlkB structure with three nucleotides. These results uncover previously unassigned key catalytic residues, identify a flexible hairpin involved in nucleotide flipping and ss/ds-DNA discrimination, and reveal self-hydroxylation of an active site leucine that may protect against uncoupled generation of dangerous oxygen radicals.