RESUMEN
B cells play a critical role in host defense, producing antibodies in response to microbial infection. An inability to produce an effective antibody response leaves affected individuals prone to serious infection; therefore, proper B-cell development is essential to human health. B-cell development begins in the bone marrow and progresses through various stages until maturation occurs in the spleen. This process involves several sequential, complex events, starting with pre- and pro-B cells, which rearrange the heavy and light chain genes responsible for producing clonally diverse immunoglobulin (Ig) molecules. These cells then differentiate into immature B cells, followed by mature B cells. The bone marrow is a complex ecological niche of supporting stromal cells, extracellular matrix components, macrophages, and hematopoietic precursor cells influencing B-cell development, maturation, and differentiation. Once fully mature, B cells circulate in peripheral lymphoid organs and can respond to antigenic stimuli. As specific cell surface markers are expressed during each stage of B-cell development, researchers use flow cytometry as a powerful tool to evaluate developmental progression. In this protocol, we provide a step-by-step method for bone marrow isolation, cell staining, and data analysis. This tool will help researchers gain a deeper understanding of the progression of B-cell development and provide a pertinent flow gating strategy.
RESUMEN
Class switch recombination generates distinct antibody isotypes critical to a robust adaptive immune system, and defects are associated with autoimmune disorders and lymphomagenesis. Transcription is required during class switch recombination to recruit the cytidine deaminase AID-an essential step for the formation of DNA double-strand breaks-and strongly induces the formation of R loops within the immunoglobulin heavy-chain locus. However, the impact of R loops on double-strand break formation and repair during class switch recombination remains unclear. Here, we report that cells lacking two enzymes involved in R loop removal-senataxin and RNase H2-exhibit increased R loop formation and genome instability at the immunoglobulin heavy-chain locus without impacting its transcriptional activity, AID recruitment, or class switch recombination efficiency. Senataxin and RNase H2-deficient cells also exhibit increased insertion mutations at switch junctions, a hallmark of alternative end joining. Importantly, these phenotypes were not observed in cells lacking senataxin or RNase H2B alone. We propose that senataxin acts redundantly with RNase H2 to mediate timely R loop removal, promoting efficient repair while suppressing AID-dependent genome instability and insertional mutagenesis.
The immune system is a complex network of cells and molecules, which helps to protect the body from invaders. The adaptive immune system can recognise millions of assailants, kill them, and 'learn' from this experience to mount an even quicker defence the next time the body is infected. To achieve this level of protection, specific immune cells, called B cells, divide when they come into contact with a molecule from a foreign particle, the antigen. The cloned B cells then produce millions of protective proteins, the antibodies, which patrol the blood stream and tag harmful particles for destruction. An antibody resembles a Y-shaped structure that contains a 'variable' region, which gives it the specificity to interact with an antigen, and a 'constant' region, which interacts with components of the immune system and determines the mechanisms used to destroy a pathogen. Based on the constant region, antibodies can be divided into five main classes. B cells are able to switch their production from one antibody class to another in an event known as class switch recombination, by making changes to the constant region. They do this by cutting out a portion of the genes for the constant region from their DNA and fusing the remaining DNA. The resulting antibodies still recognise the same target, but interact with different components of the immune system, ensuring that all the body's forces are mobilised. R-loops are temporary structures that form when a cell 'reads' the instructions in its DNA to make proteins. R-loops provide physical support by anchoring the transcription template to the DNA. They help control the activity of genes, but if they stay on the DNA for too long they could interfere with any form of. DNA repair including the cutting and fusing mechanisms during class switch recombination. To find out more about this process, Zhao et al. used B-cells from mice lacking two specific proteins that usually help to remove R-loops. Without these proteins, the B cells generated more R-loops than normal. Nevertheless, the B-cells were able to undergo class switch recombination, even though their chromosomes showed large areas of DNA damage, and DNA sections that had been repaired contained several mistakes. Errors that occur during class switch recombination have been linked to immune disorders and B cell cancers. The study of Zhao et al. shows that even if R-loops do not affect some processes in B cells, they could still impact the overall health of their DNA. A next step would be to test if an inability to remove R-loops could indeed play a role in immune disorders and B-cell cancers.
Asunto(s)
Recombinación Genética , Ribonucleasas , Humanos , Ribonucleasas/genética , Cambio de Clase de Inmunoglobulina/genética , Endorribonucleasas/genética , Isotipos de Inmunoglobulinas/genética , Inestabilidad Genómica , Citidina Desaminasa/genéticaRESUMEN
Conflicts between transcription and replication machinery are a potent source of replication stress and genome instability; however, no technique currently exists to identify endogenous genomic locations prone to transcription-replication interactions. Here, we report a novel method to identify genomic loci prone to transcription-replication interactions termed transcription-replication immunoprecipitation on nascent DNA sequencing, TRIPn-Seq. TRIPn-Seq employs the sequential immunoprecipitation of RNA polymerase 2 phosphorylated at serine 5 (RNAP2s5) followed by enrichment of nascent DNA previously labeled with bromodeoxyuridine. Using TRIPn-Seq, we mapped 1009 unique transcription-replication interactions (TRIs) in mouse primary B cells characterized by a bimodal pattern of RNAP2s5, bidirectional transcription, an enrichment of RNA:DNA hybrids, and a high probability of forming G-quadruplexes. TRIs are highly enriched at transcription start sites and map to early replicating regions. TRIs exhibit enhanced Replication Protein A association and TRI-associated genes exhibit higher replication fork termination than control transcription start sites, two marks of replication stress. TRIs colocalize with double-strand DNA breaks, are enriched for deletions, and accumulate mutations in tumors. We propose that replication stress at TRIs induces mutations potentially contributing to age-related disease, as well as tumor formation and development.
Asunto(s)
Linfocitos B/metabolismo , Replicación del ADN , Inestabilidad Genómica , Animales , Roturas del ADN de Doble Cadena , Reparación del ADN , Replicación del ADN/genética , Ratones , Transcripción GenéticaRESUMEN
Transcription-replication interactions occur when DNA replication encounters genomic regions undergoing transcription. Both replication and transcription are essential for life and use the same DNA template making conflicts unavoidable. R-loops, DNA supercoiling, DNA secondary structure, and chromatin-binding proteins are all potential obstacles for processive replication or transcription and pose an even more potent threat to genome integrity when these processes co-occur. It is critical to maintaining high fidelity and processivity of transcription and replication while navigating through a complex chromatin environment, highlighting the importance of defining cellular pathways regulating transcription-replication interaction formation, evasion, and resolution. Here we discuss how transcription influences replication fork stability, and the safeguards that have evolved to navigate transcription-replication interactions and maintain genome integrity in mammalian cells.
Asunto(s)
Cromatina/metabolismo , Replicación del ADN , Transcripción Genética , Animales , Cromosomas/metabolismo , ADN/química , Daño del ADN , ADN-Topoisomerasas de Tipo II/química , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , ARN Polimerasas Dirigidas por ADN/química , Escherichia coli , Inestabilidad Genómica , Humanos , Ratones , Conformación de Ácido Nucleico , Nucleótidos/química , Oncogenes , Unión Proteica , Reproducibilidad de los Resultados , Ribonucleasa H/química , Saccharomyces cerevisiae , Procesos EstocásticosRESUMEN
Genome instability is not only a hallmark of cancer, it is necessary for its initiation and evolution, and naturally accumulates as cells age. Replication stress is a potent source of genome instability found in many tumor types [1]. Chromosomal fragile sites are genomic loci highly prone to DNA damage specifically from replication stress and are frequently mutated in cancer [2-4]2-4]. While tracking the origin of individual mutations has proved challenging, measuring DNA damage and repair at endogenous sites can offer key insights into understanding the etiology of cancer. In the past 15 years, the causal link between replication stress, oncogene activation, and tumor initiation and evolution has become increasingly clear [1, 5-9]. Replication-associated damage accumulates at early stages of tumorigenesis and may promote further transformation. Studying the causes and consequences of fragile site instability can offer a window into the earliest stages of carcinogenesis [10-13]. In particular, fragile site studies will help us understand the molecular underpinnings influencing the frequency of DNA breakage, successful repair processes suppressing genome instability, and unsuccessful repair leading to mutations and chromosome rearrangements. Of these, measuring successful repair is the most challenging as it leaves little evidence behind.
RESUMEN
Chromosomal fragile sites are genomic loci sensitive to replication stress which accumulate high levels of DNA damage, and are frequently mutated in cancers. Fragile site damage is thought to arise from the aberrant repair of spontaneous replication stress, however successful fragile site repair cannot be calculated using existing techniques. Here, we report a new assay measuring recombination-mediated repair at endogenous genomic loci by combining a sister chromatid exchange (SCE) assay with fluorescent in situ hybridization (SCE-FISH). Using SCE-FISH, we find that endogenous and exogenous replication stress generated unrepaired breaks and SCEs at fragile sites. We also find that distinct sources of replication stress induce distinct patterns of breakage: ATR inhibition induces more breaks at early replicating fragile sites (ERFS), while ERFS and late-replicating common fragile sites (CFS) are equally fragile in response to aphidicolin. Furthermore, SCEs were suppressed at fragile sites near centromeres in response to replication stress, suggesting that genomic location influences DNA repair pathway choice. SCE-FISH also measured successful recombination in human primary lymphocytes, and identificed the proto-oncogene BCL2 as a replication stress-induced fragile site. These findings demonstrate that SCE-FISH frequency at fragile sites is a sensitive indicator of replication stress, and that large-scale genome organization influences DNA repair pathway choice.
Asunto(s)
Sitios Frágiles del Cromosoma , Daño del ADN , Reparación del ADN , Replicación del ADN , Hibridación Fluorescente in Situ/métodos , Recombinación Genética , Intercambio de Cromátides Hermanas/genética , Animales , Células Cultivadas , Proteínas de Unión al ADN/fisiología , Humanos , Linfocitos/citología , Linfocitos/metabolismo , Ratones , Ratones Noqueados , Proto-Oncogenes MasRESUMEN
In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2(TG)) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity.
Asunto(s)
Rotura Cromosómica , Sitios Frágiles del Cromosoma/genética , Dosificación de Gen/genética , Longevidad/genética , Proteínas Serina-Treonina Quinasas/genética , Ribonucleósido Difosfato Reductasa/genética , Animales , Línea Celular , Supervivencia Celular , Células Cultivadas , Activación Enzimática/genética , Fibroblastos/citología , Humanos , Ratones , Nucleósidos/metabolismo , Análisis de SupervivenciaRESUMEN
Nuclear DNA replication requires the concerted action of hundreds of proteins to efficiently unwind and duplicate the entire genome while also retaining epigenetic regulatory information. Initiation of DNA replication is tightly regulated, rapidly firing thousands of origins once the conditions to promote rapid and faithful replication are in place, and defects in replication initiation lead to proliferation defects, genome instability, and a range of developmental abnormalities. Interestingly, DNA replication in metazoans initiates in actively transcribed DNA, meaning that replication initiation occurs in DNA that is co-occupied with tens of thousands of poised and active RNA polymerase complexes. Active transcription can induce genome instability, particularly during DNA replication, as RNA polymerases can induce torsional stress, formation of secondary structures, and act as a physical barrier to other enzymes involved in DNA metabolism. Here we discuss the challenges facing mammalian DNA replication, their impact on genome instability, and the development of cancer.
Asunto(s)
Replicación del ADN , Inestabilidad Genómica , Neoplasias/genética , Origen de Réplica , Transcripción Genética , Animales , ADN/genética , Humanos , ARN/genéticaRESUMEN
DNA double-strand breaks (DSBs) in B lymphocytes arise stochastically during replication or as a result of targeted DNA damage by activation-induced cytidine deaminase (AID). Here we identify recurrent, early replicating, and AID-independent DNA lesions, termed early replication fragile sites (ERFSs), by genome-wide localization of DNA repair proteins in B cells subjected to replication stress. ERFSs colocalize with highly expressed gene clusters and are enriched for repetitive elements and CpG dinucleotides. Although distinct from late-replicating common fragile sites (CFS), the stability of ERFSs and CFSs is similarly dependent on the replication-stress response kinase ATR. ERFSs break spontaneously during replication, but their fragility is increased by hydroxyurea, ATR inhibition, or deregulated c-Myc expression. Moreover, greater than 50% of recurrent amplifications/deletions in human diffuse large B cell lymphoma map to ERFSs. In summary, we have identified a source of spontaneous DNA lesions that drives instability at preferred genomic sites.
Asunto(s)
Sitios Frágiles del Cromosoma , Replicación del ADN , Eucariontes/genética , Inestabilidad Genómica , Células Procariotas/fisiología , Animales , Fenómenos Biomecánicos , Reparación del ADN , HumanosRESUMEN
53BP1 is a DNA damage protein that forms phosphorylated H2AX (γ-H2AX) dependent foci in a 1 Mb region surrounding DNA double-strand breaks (DSBs). In addition, 53BP1 promotes genomic stability by regulating the metabolism of DNA ends. We have compared the joining rates of paired DSBs separated by 1.2 kb to 27 Mb on chromosome 12 in the presence or absence of 53BP1. 53BP1 facilitates joining of intrachromosomal DSBs but only at distances corresponding to γ-H2AX spreading. In contrast, DNA end protection by 53BP1 is distance independent. Furthermore, analysis of 53BP1 mutants shows that chromatin association, oligomerization, and N-terminal ATM phosphorylation are all required for DNA end protection and joining as measured by immunoglobulin class switch recombination. These data elucidate the molecular events that are required for 53BP1 to maintain genomic stability and point to a model wherein 53BP1 and H2AX cooperate to repress resection of DSBs.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Cambio de Clase de Inmunoglobulina/genética , Animales , Linfocitos B/metabolismo , Sitios de Unión , Western Blotting , Células Cultivadas , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Genéticos , Fosforilación , Multimerización de Proteína , Recombinación Genética , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Regulation of the repair of DNA double-strand breaks by homologous recombination is extremely important for both cell viability and the maintenance of genomic integrity. Modulation of double-strand break repair in the yeast Saccharomyces cerevisiae involves controlling the recruitment of one of the central recombination proteins, Rad52, to sites of DNA lesions. The Rad52 protein, which plays a role in strand exchange and the annealing of single strand DNA, is positively regulated upon entry into S phase, repressed during the intra-S phase checkpoint, and undergoes posttranslational modification events such as phosphorylation and sumoylation. These processes all contribute to the timing of Rad52 recruitment, its stability and function. Here, we summarize the regulatory events affecting the Rad52 protein and discuss how this regulation impacts DNA repair and cell survival.
RESUMEN
Recruitment of the homologous recombination machinery to sites of double-strand breaks is a cell cycle-regulated event requiring entry into S phase and CDK1 activity. Here, we demonstrate that the central recombination protein, Rad52, forms foci independent of DNA replication, and its recruitment requires B-type cyclin/CDK1 activity. Induction of the intra-S-phase checkpoint by hydroxyurea (HU) inhibits Rad52 focus formation in response to ionizing radiation. This inhibition is dependent upon Mec1/Tel1 kinase activity, as HU-treated cells form Rad52 foci in the presence of the PI3 kinase inhibitor caffeine. These Rad52 foci colocalize with foci formed by the replication clamp PCNA. These results indicate that Mec1 activity inhibits the recruitment of Rad52 to both sites of DNA damage and stalled replication forks during the intra-S-phase checkpoint. We propose that B-type cyclins promote the recruitment of Rad52 to sites of DNA damage, whereas Mec1 inhibits spurious recombination at stalled replication forks.
Asunto(s)
Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Replicación del ADN , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Cafeína/metabolismo , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2 , Daño del ADN , Inhibidores Enzimáticos/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Hidroxiurea/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Recombinación Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Double-strand breaks (DSBs) are potentially lethal DNA lesions that can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that DSBs induced by ionizing radiation (IR) are efficiently processed for HR and bound by Rfa1 during G1, while endonuclease-induced breaks are recognized by Rfa1 only after the cell enters S phase. This difference is dependent on the DNA end-binding Yku70/Yku80 complex. Cell-cycle regulation is also observed in the DNA damage checkpoint response. Specifically, the 9-1-1 complex is required in G1 cells to recruit the Ddc2 checkpoint protein to damaged DNA, while, upon entry into S phase, the cyclin-dependent kinase Cdc28 and the 9-1-1 complex both serve to recruit Ddc2 to foci. Together, these results demonstrate that the DNA repair machinery distinguishes between different types of damage in G1, which translates into different modes of checkpoint activation in G1 and S/G2 cells.
Asunto(s)
Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , ADN , Fase G1/fisiología , Proteínas Adaptadoras Transductoras de Señales , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN/genética , ADN/metabolismo , ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Endonucleasas , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Genes cdc , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Radiación Ionizante , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Proteína de Replicación A , Fase S/fisiología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
DNA repair is an essential process for preserving genome integrity in all organisms. In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci). Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication stress in Saccharomyces cerevisiae. The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs. Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins. Later and only in S and G2 phase, the homologous recombination machinery assembles at the site. Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse. The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.