RESUMO
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.
Assuntos
Sítios Frágeis do Cromossomo , Replicação do DNA , Eucariotos/genética , Instabilidade Genômica , Células Procarióticas/fisiologia , Animais , Fenômenos Biomecânicos , Reparo do DNA , HumanosRESUMO
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.
Assuntos
Linfócitos B/metabolismo , Replicação do DNA , Instabilidade Genômica , Animais , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA/genética , Camundongos , Transcrição GênicaRESUMO
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.
Assuntos
Quebra Cromossômica , Sítios Frágeis do Cromossomo/genética , Dosagem de Genes/genética , Longevidade/genética , Proteínas Serina-Treonina Quinases/genética , Ribonucleosídeo Difosfato Redutase/genética , Animais , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Ativação Enzimática/genética , Fibroblastos/citologia , Humanos , Camundongos , Nucleosídeos/metabolismo , Análise de SobrevidaRESUMO
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.
Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , DNA , Fase G1/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/genética , DNA/metabolismo , DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Genes cdc , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Radiação Ionizante , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Proteína de Replicação A , Fase S/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Replicação do DNA , Instabilidade Genômica , Neoplasias/genética , Origem de Replicação , Transcrição Gênica , Animais , DNA/genética , Humanos , RNA/genéticaRESUMO
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.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Replicação do DNA , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Cafeína/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Dano ao DNA , Inibidores Enzimáticos/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Hidroxiureia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Cromatina/metabolismo , Replicação do DNA , Transcrição Gênica , Animais , Cromossomos/metabolismo , DNA/química , Dano ao DNA , DNA Topoisomerases Tipo II/química , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , RNA Polimerases Dirigidas por DNA/química , Escherichia coli , Instabilidade Genômica , Humanos , Camundongos , Conformação de Ácido Nucleico , Nucleotídeos/química , Oncogenes , Ligação Proteica , Reprodutibilidade dos Testes , Ribonuclease H/química , Saccharomyces cerevisiae , Processos EstocásticosRESUMO
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.
Assuntos
Sítios Frágeis do Cromossomo , Dano ao DNA , Reparo do DNA , Replicação do DNA , Hibridização in Situ Fluorescente/métodos , Recombinação Genética , Troca de Cromátide Irmã/genética , Animais , Células Cultivadas , Proteínas de Ligação a DNA/fisiologia , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos , Camundongos Knockout , Proto-Oncogene MasRESUMO
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.
RESUMO
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.