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1.
Nat Immunol ; 25(7): 1257-1269, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38806707

RESUMO

The circadian clock is a critical regulator of immunity, and this circadian control of immune modulation has an essential function in host defense and tumor immunosurveillance. Here we use a single-cell RNA sequencing approach and a genetic model of colorectal cancer to identify clock-dependent changes to the immune landscape that control the abundance of immunosuppressive cells and consequent suppression of cytotoxic CD8+ T cells. Of these immunosuppressive cell types, PD-L1-expressing myeloid-derived suppressor cells (MDSCs) peak in abundance in a rhythmic manner. Disruption of the epithelial cell clock regulates the secretion of cytokines that promote heightened inflammation, recruitment of neutrophils and the subsequent development of MDSCs. We also show that time-of-day anti-PD-L1 delivery is most effective when synchronized with the abundance of immunosuppressive MDSCs. Collectively, these data indicate that circadian gating of tumor immunosuppression informs the timing and efficacy of immune checkpoint inhibitors.


Assuntos
Antígeno B7-H1 , Relógios Circadianos , Inibidores de Checkpoint Imunológico , Células Supressoras Mieloides , Animais , Camundongos , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Relógios Circadianos/imunologia , Antígeno B7-H1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Camundongos Endogâmicos C57BL , Ritmo Circadiano/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/terapia , Neoplasias Colorretais/tratamento farmacológico , Microambiente Tumoral/imunologia , Tolerância Imunológica , Humanos , Feminino , Linhagem Celular Tumoral , Análise de Célula Única , Terapia de Imunossupressão , Citocinas/metabolismo , Masculino
2.
Nat Rev Mol Cell Biol ; 18(8): 495-506, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28512351

RESUMO

DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage because they can result in the loss of large chromosomal regions. In all mammalian cells, DSBs that occur throughout the cell cycle are repaired predominantly by the non-homologous DNA end joining (NHEJ) pathway. Defects in NHEJ result in sensitivity to ionizing radiation and the ablation of lymphocytes. The NHEJ pathway utilizes proteins that recognize, resect, polymerize and ligate the DNA ends in a flexible manner. This flexibility permits NHEJ to function on a wide range of DNA-end configurations, with the resulting repaired DNA junctions often containing mutations. In this Review, we discuss the most recent findings regarding the relative involvement of the different NHEJ proteins in the repair of various DNA-end configurations. We also discuss the shunting of DNA-end repair to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA) and the relevance of these different pathways to human disease.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , Reparo do DNA/fisiologia , Animais , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Humanos
3.
Mol Cell ; 68(5): 901-912.e3, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29220655

RESUMO

DNA double-strand breaks (DSBs) occurring within fragile zones of less than 200 base pairs account for the formation of the most common human chromosomal translocations in lymphoid malignancies, yet the mechanism of how breaks occur remains unknown. Here, we have transferred human fragile zones into S. cerevisiae in the context of a genetic assay to understand the mechanism leading to DSBs at these sites. Our findings indicate that a combination of factors is required to sensitize these regions. Foremost, DNA strand separation by transcription or increased torsional stress can expose these DNA regions to damage from either the expression of human AID or increased oxidative stress. This damage causes DNA lesions that, if not repaired quickly, are prone to nuclease cleavage, resulting in DSBs. Our results provide mechanistic insight into why human neoplastic translocation fragile DNA sequences are more prone to enzymes or agents that cause longer-lived DNA lesions.


Assuntos
Cromossomos Humanos/genética , Citidina Desaminase/genética , Quebras de DNA de Cadeia Dupla , DNA Fúngico/genética , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Translocação Genética , Cromossomos Humanos/química , Cromossomos Humanos/metabolismo , Citidina Desaminase/metabolismo , DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA , Endonucleases/genética , Endonucleases/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Peroxidases/genética , Peroxidases/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Transcrição Gênica , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo
4.
Eur J Haematol ; 112(3): 392-401, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37933194

RESUMO

OBJECTIVE: There are significant disparities in outcomes among Hispanic patients with acute lymphoblastic leukemia (ALL). Recent studies have demonstrated favorable outcomes of pegaspargase-containing ALL regimens (PEG-CAR) in young adults however, outcomes in Hispanic ethnicity continue to be underreported. METHODS: We evaluated outcomes of newly diagnosed, adult B-cell ALL Hispanic and non-Hispanic patients consecutively treated with a PEG-CAR or HyperCVAD between January 2011 and November 2022. The primary endpoint was event-free survival (EFS) while secondary endpoints included cumulative incidence of relapse and overall survival (OS). RESULTS: Among 105 included patients, 48 (45.7%) were treated with a PEG-CAR and 57 (54.3%) with HyperCVAD. Median age was 38 years (range, 18-75 years), 61% were Hispanic, and 35.2% had poor-genetic risk. Hispanic patients demonstrated significantly worse 5-year EFS with a PEG-CAR compared to that seen with HyperCVAD (HR, 2.58; 95% CI, 1.32-5.04; p = .006) whereas non-Hispanic patients had better outcomes with PIR (52.4% vs. 42.0%). Hispanic ethnicity (p = .015) and male sex (p = .019) were independent predictors for poor OS. CONCLUSIONS: Hispanic patients with B-cell ALL had worse EFS with a PEG-CAR as compared with HyperCVAD. Future studies will aim to confirm these findings and establish a tailored treatment approach for this high-risk population.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adulto Jovem , Humanos , Masculino , Adulto , Asparaginase/efeitos adversos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiologia , Polietilenoglicóis/efeitos adversos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Estudos Retrospectivos
5.
Mol Cell ; 62(3): 327-334, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-27153532

RESUMO

The twisting of DNA due to the movement of RNA polymerases is the basis of numerous classic experiments in molecular biology. Recent mouse genetic models indicate that chromosomal breakage is common at sites of transcriptional turbulence. Two key studies on this point mapped breakpoints to sites of either convergent or divergent transcription but arrived at different conclusions as to which is more detrimental and why. The issue hinges on whether DNA strand separation is the basis for the chromosomal instability or collision of RNA polymerases.


Assuntos
Instabilidade Cromossômica , Dano ao DNA , RNA Polimerases Dirigidas por DNA/metabolismo , DNA/metabolismo , Transcrição Gênica , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , DNA/química , DNA Bacteriano/química , DNA Bacteriano/metabolismo , DNA Fúngico/química , DNA Fúngico/metabolismo , Humanos , Modelos Genéticos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Conformação de Ácido Nucleico
6.
Trends Biochem Sci ; 43(7): 490-498, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29735400

RESUMO

A subset of chromosomal translocations related to B cell malignancy in human patients arises due to DNA breaks occurring within defined 20-600 base pair (bp) zones. Several factors influence the breakage rate at these sites including transcription, DNA sequence, and topological tension. These factors favor non-B DNA structures that permit formation of transient single-stranded DNA (ssDNA), making the DNA more vulnerable to agents such as the enzyme activation-induced cytidine deaminase (AID) and reactive oxygen species (ROS). Certain DNA lesions created during the ssDNA state persist after the DNA resumes its normal duplex structure. We propose that factors favoring both formation of transient ssDNA and persistent DNA lesions are key in determining the DNA breakage mechanism.


Assuntos
Sítios Frágeis do Cromossomo , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Leucemia de Células B/genética , Modelos Genéticos , Translocação Genética , Animais , Citidina Desaminase/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Humanos , Cinética , Leucemia de Células B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie
8.
J Biol Chem ; 293(27): 10512-10523, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29247009

RESUMO

Nonhomologous DNA end-joining (NHEJ) is the predominant double-strand break (DSB) repair pathway throughout the cell cycle and accounts for nearly all DSB repair outside of the S and G2 phases. NHEJ relies on Ku to thread onto DNA termini and thereby improve the affinity of the NHEJ enzymatic components consisting of polymerases (Pol µ and Pol λ), a nuclease (the Artemis·DNA-PKcs complex), and a ligase (XLF·XRCC4·Lig4 complex). Each of the enzymatic components is distinctive for its versatility in acting on diverse incompatible DNA end configurations coupled with a flexibility in loading order, resulting in many possible junctional outcomes from one DSB. DNA ends can either be directly ligated or, if the ends are incompatible, processed until a ligatable configuration is achieved that is often stabilized by up to 4 bp of terminal microhomology. Processing of DNA ends results in nucleotide loss or addition, explaining why DSBs repaired by NHEJ are rarely restored to their original DNA sequence. Thus, NHEJ is a single pathway with multiple enzymes at its disposal to repair DSBs, resulting in a diversity of repair outcomes.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Autoantígeno Ku/metabolismo , Animais , Humanos
9.
Nucleic Acids Res ; 42(21): 13186-93, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25378327

RESUMO

The boundaries of R-loops are well-documented at immunoglobulin heavy chain loci in mammalian B cells. Within primary B cells or B cell lines, the upstream boundaries of R-loops typically begin early in the repetitive portion of the switch regions. Most R-loops terminate within the switch repetitive zone, but the remainder can extend a few hundred base pairs further, where G-density on the non-template DNA strand gradually drops to the genome average. Whether the G-density determines how far the R-loops extend is an important question. We previously studied the role of G-clusters in initiating R-loop formation, but we did not examine the role of G-density in permitting the elongation of the R-loop, after it had initiated. Here, we vary the G-density of different portions of the switch region in a murine B cell line. We find that both class switch recombination (CSR) and R-loop formation decrease significantly when the overall G-density is reduced from 46% to 29%. Short 50 bp insertions with low G-density within switch regions do not appear to affect either CSR or R-loop elongation, whereas a longer (150 bp) insertion impairs both. These results demonstrate that G-density is an important determinant of the length over which mammalian genomic R-loops extend.


Assuntos
Switching de Imunoglobulina , Região de Troca de Imunoglobulinas , Animais , Linhagem Celular , DNA/química , Camundongos , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico
10.
Sci Adv ; 10(39): eado1458, 2024 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-39331712

RESUMO

Diet is a robust entrainment cue that regulates diurnal rhythms of the gut microbiome. We and others have shown that disruption of the circadian clock drives the progression of colorectal cancer (CRC). While certain bacterial species have been suggested to play driver roles in CRC, it is unknown whether the intestinal clock impinges on the microbiome to accelerate CRC pathogenesis. To address this, genetic disruption of the circadian clock, in an Apc-driven mouse model of CRC, was used to define the impact on the gut microbiome. When clock disruption is combined with CRC, metagenomic sequencing identified dysregulation of many bacterial genera including Bacteroides, Helicobacter, and Megasphaera. We identify functional changes to microbial pathways including dysregulated nucleic acid, amino acid, and carbohydrate metabolism, as well as disruption of intestinal barrier function. Our findings suggest that clock disruption impinges on microbiota composition and intestinal permeability that may contribute to CRC pathogenesis.


Assuntos
Relógios Circadianos , Neoplasias Colorretais , Disbiose , Microbioma Gastrointestinal , Animais , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Disbiose/microbiologia , Camundongos , Relógios Circadianos/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Modelos Animais de Doenças , Humanos , Permeabilidade
11.
Nat Commun ; 15(1): 4634, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38821984

RESUMO

The master DNA damage repair histone protein, H2AX, is essential for orchestrating the recruitment of downstream mediator and effector proteins at damaged chromatin. The phosphorylation of H2AX at S139, γH2AX, is well-studied for its DNA repair function. However, the extended C-terminal tail is not characterized. Here, we define the minimal motif on H2AX for the canonical function in activating the MDC1-RNF8-RNF168 phosphorylation-ubiquitination pathway that is important for recruiting repair proteins, such as 53BP1 and BRCA1. Interestingly, H2AX recruits 53BP1 independently from the MDC1-RNF8-RNF168 pathway through its evolved C-terminal linker region with S139 phosphorylation. Mechanistically, 53BP1 recruitment to damaged chromatin is mediated by the interaction between the H2AX C-terminal tail and the 53BP1 Oligomerization-Tudor domains. Moreover, γH2AX-linker mediated 53BP1 recruitment leads to camptothecin resistance in H2AX knockout cells. Overall, our study uncovers an evolved mechanism within the H2AX C-terminal tail for regulating DNA repair proteins at damaged chromatin.


Assuntos
Cromatina , Dano ao DNA , Histonas , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitinação , Humanos , Proteínas Adaptadoras de Transdução de Sinal , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Camptotecina/farmacologia , Proteínas de Ciclo Celular , Cromatina/metabolismo , Reparo do DNA , Células HEK293 , Histonas/metabolismo , Histonas/genética , Fosforilação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética
12.
Nat Commun ; 15(1): 6331, 2024 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-39068148

RESUMO

Activation-induced cytidine deaminase (AID) is a B cell-specific mutator required for antibody diversification. However, it is also implicated in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain blood cancers is critical in assessing disease severity and treatment options. We have developed a digital PCR (dPCR) assay that allows us to quantify mutations resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this assay shows that increased AID levels in immature B cells increase genome instability at loci linked to chromosomal translocation formation. This includes the CRLF2 locus that is often involved in translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Hispanics, particularly those with Latin American ancestry. Using dPCR, we characterize the CRLF2 locus in B cell-derived genomic DNA from both Hispanic ALL patients and healthy Hispanic donors and found increased mutations in both, suggesting that vulnerability to DNA damage at CRLF2 may be driving this health disparity. Our ability to detect and quantify these mutations will potentiate future risk identification, early detection of cancers, and reduction of associated cancer health disparities.


Assuntos
Citidina Desaminase , Hispânico ou Latino , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Citocinas , Humanos , Citidina Desaminase/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Hispânico ou Latino/genética , Receptores de Citocinas/genética , Quebras de DNA de Cadeia Dupla , Linfócitos B/metabolismo , Linfócitos B/imunologia , Disparidades nos Níveis de Saúde , Translocação Genética , Loci Gênicos , América Latina , Feminino
13.
Elife ; 122024 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-38224289

RESUMO

Inter-organ communication is a vital process to maintain physiologic homeostasis, and its dysregulation contributes to many human diseases. Given that circulating bioactive factors are stable in serum, occur naturally, and are easily assayed from blood, they present obvious focal molecules for therapeutic intervention and biomarker development. Recently, studies have shown that secreted proteins mediating inter-tissue signaling could be identified by 'brute force' surveys of all genes within RNA-sequencing measures across tissues within a population. Expanding on this intuition, we reasoned that parallel strategies could be used to understand how individual genes mediate signaling across metabolic tissues through correlative analyses of gene variation between individuals. Thus, comparison of quantitative levels of gene expression relationships between organs in a population could aid in understanding cross-organ signaling. Here, we surveyed gene-gene correlation structure across 18 metabolic tissues in 310 human individuals and 7 tissues in 103 diverse strains of mice fed a normal chow or high-fat/high-sucrose (HFHS) diet. Variation of genes such as FGF21, ADIPOQ, GCG, and IL6 showed enrichments which recapitulate experimental observations. Further, similar analyses were applied to explore both within-tissue signaling mechanisms (liver PCSK9) and genes encoding enzymes producing metabolites (adipose PNPLA2), where inter-individual correlation structure aligned with known roles for these critical metabolic pathways. Examination of sex hormone receptor correlations in mice highlighted the difference of tissue-specific variation in relationships with metabolic traits. We refer to this resource as gene-derived correlations across tissues (GD-CAT) where all tools and data are built into a web portal enabling users to perform these analyses without a single line of code (gdcat.org). This resource enables querying of any gene in any tissue to find correlated patterns of genes, cell types, pathways, and network architectures across metabolic organs.


Assuntos
Pró-Proteína Convertase 9 , Transdução de Sinais , Humanos , Animais , Camundongos , Homeostase , Adiposidade
14.
F1000Res ; 12: 116, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-39282509

RESUMO

In modern society, there is a growing population affected by circadian clock disruption through night shift work, artificial light-at-night exposure, and erratic eating patterns. Concurrently, the rate of cancer incidence in individuals under the age of 50 is increasing at an alarming rate, and though the precise risk factors remain undefined, the potential links between circadian clock deregulation and young-onset cancers is compelling. To explore the complex biological functions of the clock, this review will first provide a framework for the mammalian circadian clock in regulating critical cellular processes including cell cycle control, DNA damage response, DNA repair, and immunity under conditions of physiological homeostasis. Additionally, this review will deconvolute the role of the circadian clock in cancer, citing divergent evidence suggesting tissue-specific roles of the biological pacemaker in cancer types such as breast, lung, colorectal, and hepatocellular carcinoma. Recent evidence has emerged regarding the role of the clock in the intestinal epithelium, as well as new insights into how genetic and environmental disruption of the clock is linked with colorectal cancer, and the molecular underpinnings of these findings will be discussed. To place these findings within a context and framework that can be applied towards human health, a focus on how the circadian clock can be leveraged for cancer prevention and chronomedicine-based therapies will be outlined.


Assuntos
Relógios Circadianos , Neoplasias , Humanos , Relógios Circadianos/fisiologia , Animais , Ritmo Circadiano/fisiologia
15.
Res Sq ; 2023 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-37790327

RESUMO

Activation-induced cytidine deaminase (AID) is a B cell-specific base editor required during class switch recombination and somatic hypermutation for B cell maturation and antibody diversification. However, it has also been implicated as a factor in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain types of blood cancers is critical in assessing disease severity and treatment options. Here, we have developed a digital PCR (dPCR) assay that allows us to track the mutational landscape resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this new assay showed that increased AID levels in immature B cells increases genome instability at loci linked to translocation formation. This included the CRLF2 locus that is often involved in chromosomal translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Latin Americans (LAs). To support this LA-specific identification of AID mutation signatures, we characterized DNA from immature B cells isolated from the bone marrow of ALL patients. Our ability to detect and quantify these mutation signatures will potentiate future risk identification, early detection of cancers, and reduction of associated cancer health disparities.

17.
Trends Cancer ; 8(4): 328-343, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35094960

RESUMO

Chromosomal translocations arising from aberrant repair of multiple DNA double-strand breaks (DSBs) are a defining characteristic of many cancers. DSBs are an essential part of physiological processes in antibody-producing B cells. The B cell environment is poised to generate genome instability leading to translocations relevant to the pathology of blood cancers. These are a diverse set of cancers, but limited data from under-represented groups have pointed to health disparities associated with each. We focus on the DSBs that occur in developing B cells and propose the most likely mechanism behind the formation of translocations. We also highlight specific cancers in which these rearrangements occur and address the growing concern of health disparities associated with them.


Assuntos
Quebras de DNA de Cadeia Dupla , Neoplasias , DNA , Reparo do DNA por Junção de Extremidades , Reparo do DNA/genética , Humanos , Neoplasias/genética
18.
Sci Adv ; 8(32): eabo2389, 2022 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-35947664

RESUMO

An alarming rise in young onset colorectal cancer (CRC) has been reported; however, the underlying molecular mechanism remains undefined. Suspected risk factors of young onset CRC include environmental aspects, such as lifestyle and dietary factors, which are known to affect the circadian clock. We find that both genetic disruption and environmental disruption of the circadian clock accelerate Apc-driven CRC pathogenesis in vivo. Using an intestinal organoid model, we demonstrate that clock disruption promotes transformation by driving Apc loss of heterozygosity, which hyperactivates Wnt signaling. This up-regulates c-Myc, a known Wnt target, which drives heightened glycolytic metabolism. Using patient-derived organoids, we show that circadian rhythms are lost in human tumors. Last, we identify that variance between core clock and Wnt pathway genes significantly predicts the survival of patients with CRC. Overall, our findings demonstrate a previously unidentified mechanistic link between clock disruption and CRC, which has important implications for young onset cancer prevention.


Assuntos
Relógios Circadianos , Neoplasias Colorretais , Relógios Circadianos/genética , Ritmo Circadiano/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Humanos , Perda de Heterozigosidade , Organoides/metabolismo , Via de Sinalização Wnt
19.
Curr Genet ; 56(1): 87-100, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20012294

RESUMO

Studies in the budding yeast, Saccharomyces cerevisiae, have demonstrated that a substantial fraction of double-strand break repair following acute radiation exposure involves homologous recombination between repetitive genomic elements. We have previously described an assay in S. cerevisiae that allows us to model how repair of multiple breaks leads to the formation of chromosomal translocations by single-strand annealing (SSA) and found that Rad59, a paralog of the single-stranded DNA annealing protein Rad52, is critically important in this process. We have constructed several rad59 missense alleles to study its function more closely. Characterization of these mutants revealed proportional defects in both translocation formation and spontaneous direct-repeat recombination, which is also thought to occur by SSA. Combining the rad59 missense alleles with a null allele of RAD1, which encodes a subunit of a nuclease required for the removal of non-homologous tails from annealed intermediates, substantially suppressed the low frequency of translocations observed in rad1-null single mutants. These data suggest that at least one role of Rad59 in translocation formation by SSA is supporting the machinery required for cleavage of non-homologous tails.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Rad51 Recombinase/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Translocação Genética , Alelos , Quebras de DNA de Cadeia Dupla , Mutação de Sentido Incorreto , Recombinação Genética
20.
DNA Repair (Amst) ; 7(5): 788-800, 2008 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-18373960

RESUMO

Exposure to ionizing radiation results in a variety of genome rearrangements that have been linked to tumor formation. Many of these rearrangements are thought to arise from the repair of double-strand breaks (DSBs) by several mechanisms, including homologous recombination (HR) between repetitive sequences dispersed throughout the genome. Doses of radiation sufficient to create DSBs in or near multiple repetitive elements simultaneously could initiate single-strand annealing (SSA), a highly efficient, though mutagenic, mode of DSB repair. We have investigated the genetic control of the formation of translocations that occur spontaneously and those that form after the generation of DSBs adjacent to homologous sequences on two, non-homologous chromosomes in Saccharomyces cerevisiae. We found that mutations in a variety of DNA repair genes have distinct effects on break-stimulated translocation. Furthermore, the genetic requirements for repair using 300bp and 60bp recombination substrates were different, suggesting that the SSA apparatus may be altered in response to changing substrate lengths. Notably, RAD59 was found to play a particularly significant role in recombination between the short substrates that was partially independent of that of RAD52. The high frequency of these events suggests that SSA may be an important mechanism of genome rearrangement following acute radiation exposure.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Translocação Genética/genética , Southern Blotting , Diploide , Recombinação Genética
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