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1.
Science ; 377(6612): eabj5502, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-36108018

RESUMO

Chromosomal translocations result from the joining of DNA double-strand breaks (DSBs) and frequently cause cancer. However, the steps linking DSB formation to DSB ligation remain undeciphered. We report that DNA replication timing (RT) directly regulates lymphomagenic Myc translocations during antibody maturation in B cells downstream of DSBs and independently of DSB frequency. Depletion of minichromosome maintenance complexes alters replication origin activity, decreases translocations, and deregulates global RT. Ablating a single origin at Myc causes an early-to-late RT switch, loss of translocations, and reduced proximity with the immunoglobulin heavy chain (Igh) gene, its major translocation partner. These phenotypes were reversed by restoring early RT. Disruption of early RT also reduced tumorigenic translocations in human leukemic cells. Thus, RT constitutes a general mechanism in translocation biogenesis linking DSB formation to DSB ligation.


Assuntos
Período de Replicação do DNA , Translocação Genética , Carcinogênese/genética , DNA , Quebras de DNA de Cadeia Dupla , Humanos , Cadeias Pesadas de Imunoglobulinas/genética
2.
FASEB J ; 36(10): e22545, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36094323

RESUMO

The kidneys are radiosensitive and dose-limiting organs for radiotherapy (RT) targeting abdominal and paraspinal tumors. Excessive radiation doses to the kidneys ultimately lead to radiation nephropathy. Our prior work unmasked a novel role for the lipid-modifying enzyme, sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b), in regulating the response of renal podocytes to radiation injury. In this study, we investigated the role of SMPDL3b in DNA double-strand breaks (DSBs) repair in vitro and in vivo. We assessed the kinetics of DSBs recognition and repair along with the ATM pathway and nuclear sphingolipid metabolism in wild-type (WT) and SMPDL3b overexpressing (OE) human podocytes. We also assessed the extent of DNA damage repair in SMPDL3b knock-down (KD) human podocytes, and C57BL6 WT and podocyte-specific SMPDL3b-knock out (KO) mice after radiation injury. We found that SMPDL3b overexpression enhanced DSBs recognition and repair through modulating ATM nuclear shuttling. OE podocytes were protected against radiation-induced apoptosis by increasing the phosphorylation of p53 at serine 15 and attenuating subsequent caspase-3 cleavage. SMPDL3b overexpression prevented radiation-induced alterations in nuclear ceramide-1-phosphate (C1P) and ceramide levels. Interestingly, exogenous C1P pretreatment radiosensitized OE podocytes by delaying ATM nuclear foci formation and DSBs repair. On the other hand, SMPDL3b knock-down, in vitro and in vivo, induced a significant delay in DSBs repair. Additionally, increased activation of apoptosis was induced in podocytes of SMPDL3b-KO mice compared to WT mice at 24 h post-irradiation. Together, our results unravel a novel role for SMPDL3b in radiation-induced DNA damage response. The current work suggests that SMPDL3b modulates nuclear sphingolipid metabolism, ATM nuclear shuttling, and DSBs repair.


Assuntos
Podócitos , Lesões por Radiação , Animais , Ceramidas/metabolismo , Quebras de DNA de Cadeia Dupla , Humanos , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Podócitos/metabolismo , Lesões por Radiação/genética , Lesões por Radiação/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo
3.
Genome Biol ; 23(1): 187, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36068616

RESUMO

BACKGROUND: The PRDM9-dependent histone methylation H3K4me3 and H3K36me3 function in assuring accurate homologous recombination at recombination hotspots in mammals. Beyond histone methylation, H3 lysine 9 acetylation (H3K9ac) is also greatly enriched at recombination hotspots. Previous work has indicated the potential cross-talk between H3K4me3 and H3K9ac at recombination hotspots, but it is still unknown what molecular mechanisms mediate the cross-talk between the two histone modifications at hotspots or how the cross-talk regulates homologous recombination in meiosis. RESULTS: Here, we find that the histone methylation reader ZCWPW1 is essential for maintaining H3K9ac by antagonizing HDAC proteins' deacetylation activity and further promotes chromatin openness at recombination hotspots thus preparing the way for homologous recombination during meiotic double-strand break repair. Interestingly, ectopic expression of the germ-cell-specific protein ZCWPW1 in human somatic cells enhances double-strand break repair via homologous recombination. CONCLUSIONS: Taken together, our findings provide new insights into how histone modifications and their associated regulatory proteins collectively regulate meiotic homologous recombination.


Assuntos
Cromatina , Código das Histonas , Animais , Quebras de DNA de Cadeia Dupla , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Recombinação Homóloga , Humanos , Mamíferos/metabolismo , Meiose
4.
Int J Mol Sci ; 23(17)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36077210

RESUMO

Meiosis initiates with the formation of double strand breaks (DSBs) throughout the genome. To avoid genomic instability, these DSBs need to be correctly repaired by homologous recombination. Surveillance mechanisms involving the DNA damage response (DDR) pathway ATM-CHK2-p53 can detect the persistence of unrepaired DBSs and activate the recombination-dependent arrest at the pachytene stage. However, a complete understanding of p53 functions under normal physiological conditions remains lacking. Here, we report a detailed analysis of the p53 role during meiotic prophase in mice spermatocytes. We show that the absence of p53 regulates prophase progression by slowing down the pachytene stage when the recombination-dependent arrest occurs. Furthermore, our results show that p53 is necessary for proper crossover (CO) formation and localization. Our study contributes to a deeper understanding of p53 roles during the meiotic prophase.


Assuntos
Quebras de DNA de Cadeia Dupla , Meiose , Animais , Proteínas de Ciclo Celular/metabolismo , Masculino , Camundongos , Prófase , Espermatócitos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
5.
Int J Mol Sci ; 23(17)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36077220

RESUMO

Topoisomerase inhibitors are widely used in cancer chemotherapy. However, one of the potential long-term adverse effects of such therapy is acute leukemia. A key feature of such therapy-induced acute myeloid leukemia (t-AML) is recurrent chromosomal translocations involving AML1 (RUNX1) or MLL (KMT2A) genes. The formation of chromosomal translocation depends on the spatial proximity of translocation partners and the mobility of the DNA ends. It is unclear which of these two factors might be decisive for recurrent t-AML translocations. Here, we used fluorescence in situ hybridization (FISH) and chromosome conformation capture followed by sequencing (4C-seq) to investigate double-strand DNA break formation and the mobility of broken ends upon etoposide treatment, as well as contacts between translocation partner genes. We detected the separation of the parts of the broken AML1 gene, as well as the increased mobility of these separated parts. 4C-seq analysis showed no evident contacts of AML1 and MLL with loci, implicated in recurrent t-AML translocations, either before or after etoposide treatment. We suggest that separation of the break ends and their increased non-targeted mobility-but not spatial predisposition of the rearrangement partners-plays a major role in the formation of these translocations.


Assuntos
Leucemia Mieloide Aguda , Translocação Genética , DNA , Quebras de DNA de Cadeia Dupla , Etoposídeo/efeitos adversos , Humanos , Hibridização in Situ Fluorescente , Leucemia Mieloide Aguda/genética , Inibidores da Topoisomerase II/efeitos adversos
6.
Int J Mol Sci ; 23(17)2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36077304

RESUMO

Meiotic recombination plays a pivotal role in achieving accurate chromosomal segregation and increasing genetic diversity. In the homologous recombination pathway, the detailed mechanisms of how OsRAD51 and OsDMC1 work in rice meiosis remain to be explored. Here, we obtained different types of mutants for Osrad51a1, Osrad51a2, Osdmc1a, and Osdmc1b through CRISPR/Cas9. Both Osrad51a1 and Osrad51a2 exhibited normal vegetative growth and fertility. Osrad51 (Osrad51a1 Osrad51a2) mutant plants show normal vegetative growth but exhibit complete sterility, indicating that OsRAD51A1 and OsRAD51A2 are functionally redundant in rice fertility. In contrast to the wild type, Osrad51 chromosomes are not paired perfectly at pachytene and synaptonemal complex (SC) formation is deficient. Moreover, univalents and multivalent associations were observed at metaphase I, chromosome fragments presented at anaphase I, and crossover formation is basically suppressed in Osrad51 pollen mother cells (PMCs). OsRAD51 foci emerge at leptotene and disappear from late pachytene and chromosome localization of OsRAD51 depends on the formation of double-strand breaks (DSBs). Most OsRAD51 foci can co-localize with OsDMC1 signals. OsRAD51 is essential for the loading of OsDMC1 onto chromosomes, and vice versa. In addition, both OsRAD51 and OsDMC1 can interact with OsFIGL1 and OsBRCA2, two important components in rice meiosis. Moreover, the Osrad51 Osdmc1 (Osrad51a1 Osrad51a2 Osdmc1a Osdmc1b) quadruple mutant PMCs exhibited similar defective phenotypes as Osrad51 in homologous pairing, synapsis, and DSB repair. Taken together, our results suggest that the recombinases DMC1 and RAD51 may functionally depend on each other and play important roles in meiotic recombination during meiosis in rice.


Assuntos
Oryza , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Meiose/genética , Oryza/genética , Oryza/metabolismo
7.
Int J Mol Sci ; 23(17)2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36077590

RESUMO

53BP1 protein has been isolated in-vitro as a putative p53 interactor. From the discovery of its engagement in the DNA-Damage Response (DDR), its role in sustaining the activity of the p53-regulated transcriptional program has been frequently under-evaluated, even in the case of a specific response to numerous DNA Double-Strand Breaks (DSBs), i.e., exposure to ionizing radiation. The localization of 53BP1 protein constitutes a key to decipher the network of activities exerted in response to stress. We present here an automated-microscopy for image cytometry protocol to analyze the evolution of the DDR, and to demonstrate how 53BP1 moved from damaged sites, where the well-known interaction with the DSB marker γH2A.X takes place, to nucleoplasm, interacting with p53, and enhancing the transcriptional regulation of the guardian of the genome protein. Molecular interactions have been quantitatively described and spatiotemporally localized at the highest spatial resolution by a simultaneous analysis of the impairment of the cell-cycle progression. Thanks to the high statistical sampling of the presented protocol, we provide a detailed quantitative description of the molecular events following the DSBs formation. Single-Molecule Localization Microscopy (SMLM) Analysis finally confirmed the p53-53BP1 interaction on the tens of nanometers scale during the distinct phases of the response.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteína Supressora de Tumor p53 , DNA/metabolismo , Dano ao DNA , Reparo do DNA , Citometria por Imagem , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
8.
Radiat Prot Dosimetry ; 198(13-15): 1009-1013, 2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-36083726

RESUMO

To evaluate biological effects triggered by low levels of radiation, we established a uniquely sensitive experimental system to detect somatic mutations. By using the system, we found that mutant frequencies induced by X-rays were statistically significant at doses over 0.15 Gy, and a linear dose relationship with the mutant frequency was observed at doses over 0.15 Gy. The mutation spectra analysis revealed that mutation events generated by X-ray doses below 0.1 Gy were similar to those observed in unirradiated controls. In addition, a significant inflection point for both, the mutant frequency and the mutation spectra, was found at dose-rates around 11 mGy/day when cells were cultured in medium containing tritiated water. Because induced radiation-type events presented a clear dose/dose-rate dependency above the critical dose or the inflection point, these observations suggest that mutation events generated by radiation could change at a threshold dose-rate or a critical dose.


Assuntos
Partículas beta , Quebras de DNA de Cadeia Dupla , Relação Dose-Resposta à Radiação , Mutação , Trítio , Raios X
9.
Radiat Prot Dosimetry ; 198(13-15): 990-997, 2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-36083749

RESUMO

It is generally and widely accepted that the biological effects of a given dose of ionizing radiation, especially those of low linear energy transfer radiations like X-ray and gamma ray, become smaller as the dose rate becomes lower. This phenomenon, known as 'dose-rate effect (DRE),' is considered due to the repair of sublethal damage during irradiation but the precise mechanisms for DRE have remained to be clarified. We recently showed that DRE in terms of clonogenic cell survival is diminished or even inversed in rodent cells lacking Ku, which is one of the essential factors in the repair of DNA double-strand breaks (DSBs) through non-homologous end joining (NHEJ). Here we review and discuss the involvement of NHEJ in DRE, which has potential implications in radiological protection and cancer therapeutics.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA , Reparo do DNA , Transferência Linear de Energia
10.
Nat Commun ; 13(1): 5303, 2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-36085345

RESUMO

The RNA world is changing our views about sensing and resolution of DNA damage. Here, we develop single-molecule DNA/RNA analysis approaches to visualize how nascent RNA facilitates the repair of DNA double-strand breaks (DSBs). RNA polymerase II (RNAPII) is crucial for DSB resolution in human cells. DSB-flanking, RNAPII-generated nascent RNA forms RNA:DNA hybrids, guiding the upstream DNA repair steps towards favouring the error-free Homologous Recombination (HR) pathway over Non-Homologous End Joining. Specific RNAPII inhibitor, THZ1, impairs recruitment of essential HR proteins to DSBs, implicating nascent RNA in DNA end resection, initiation and execution of HR repair. We further propose that resection factor CtIP interacts with and helps re-activate RNAPII when paused by the RNA:DNA hybrids, collectively promoting faithful repair of chromosome breaks to maintain genomic integrity.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Reparo do DNA por Junção de Extremidades , Humanos , RNA/genética , RNA Polimerase II , Reparo de DNA por Recombinação
11.
Elife ; 112022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36125964

RESUMO

Repair of DNA double-strand breaks (DSBs) is crucial for genome integrity. A conserved response to DSBs is an increase in chromatin mobility that can be local, at the site of the DSB, or global, at undamaged regions of the genome. Here, we address the function of global chromatin mobility during homologous recombination (HR) of a single, targeted, controlled DSB. We set up a system that tracks HR in vivo over time and show that two types of DSB-induced global chromatin mobility are involved in HR, depending on the position of the DSB. Close to the centromere, a DSB induces global mobility that depends solely on H2A(X) phosphorylation and accelerates repair kinetics, but is not essential. In contrast, the global mobility induced by a DSB away from the centromere becomes essential for HR repair and is triggered by homology search through a mechanism that depends on H2A(X) phosphorylation, checkpoint progression, and Rad51. Our data demonstrate that global mobility is governed by chromosomal conformation and differentially coordinates repair by HR.


Assuntos
Cromatina , Quebras de DNA de Cadeia Dupla , Cromossomos , DNA , Recombinação Homóloga
12.
Nat Commun ; 13(1): 5167, 2022 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-36075897

RESUMO

Protection of stalled replication forks is essential to prevent genome instability, a major driving force of tumorigenesis. Several key regulators of DNA double-stranded break (DSB) repair, including 53BP1 and RIF1, have been implicated in fork protection. MAD2L2, also known as REV7, plays an important role downstream of 53BP1/RIF1 by counteracting resection at DSBs in the recently discovered shieldin complex. The ability to bind and counteract resection at exposed DNA ends at DSBs makes MAD2L2/shieldin a prime candidate for also suppressing nucleolytic processing at stalled replication forks. However, the function of MAD2L2/shieldin outside of DNA repair is unknown. Here we address this by using genetic and single-molecule analyses and find that MAD2L2 is required for protecting and restarting stalled replication forks. MAD2L2 loss leads to uncontrolled MRE11-dependent resection of stalled forks and single-stranded DNA accumulation, which causes irreparable genomic damage. Unexpectedly, MAD2L2 limits resection at stalled forks independently of shieldin, since fork protection remained unaffected by shieldin loss. Instead, MAD2L2 cooperates with the DNA polymerases REV3L and REV1 to promote fork stability. Thus, MAD2L2 suppresses aberrant nucleolytic processing both at DSBs and stalled replication forks by differentially engaging shieldin and REV1/REV3L, respectively.


Assuntos
Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Instabilidade Genômica , Humanos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo
13.
Nat Commun ; 13(1): 5295, 2022 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-36075911

RESUMO

DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to mutagenesis and genomic loss. DSB-repair pathway cross-talk and compensation is incompletely understood, despite its importance for genomic stability, oncogenesis, and genome editing using CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple DNA repair pathways at a DSB. DSB-Spectrum reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibiting the c-NHEJ factor DNA-PKcs increases repair by HR, but also substantially increases mutagenic SSA. Our data indicate that SSA-mediated DSB-repair also occurs at endogenous genomic loci, driven by Alu elements or homologous gene regions. Finally, we demonstrate that long-range end-resection factors DNA2 and Exo1 promote SSA and reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.


Assuntos
Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Sistemas CRISPR-Cas/genética , DNA , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Recombinação Homóloga
14.
Nat Commun ; 13(1): 5133, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36050397

RESUMO

DNA end resection is delicately regulated through various types of post-translational modifications to initiate homologous recombination, but the involvement of SUMOylation in this process remains incompletely understood. Here, we show that MRE11 requires SUMOylation to shield it from ubiquitin-mediated degradation when resecting damaged chromatin. Upon DSB induction, PIAS1 promotes MRE11 SUMOylation on chromatin to initiate DNA end resection. Then, MRE11 is deSUMOylated by SENP3 mainly after it has moved away from DSB sites. SENP3 deficiency results in MRE11 degradation failure and accumulation on chromatin, causing genome instability. We further show that cancer-related MRE11 mutants with impaired SUMOylation exhibit compromised DNA repair ability. Thus, we demonstrate that MRE11 SUMOylation in coordination with ubiquitylation is dynamically controlled by PIAS1 and SENP3 to facilitate DNA end resection and maintain genome stability.


Assuntos
Cromatina , Sumoilação , Cisteína Endopeptidases/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Instabilidade Genômica , Homeostase , Humanos , Ubiquitinação
15.
Cell Death Dis ; 13(9): 754, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36050295

RESUMO

Ivermectin is a widely used antiparasitic drug and shows promising anticancer activity in various cancer types. Although multiple signaling pathways modulated by ivermectin have been identified in tumor cells, few studies have focused on the exact target of ivermectin. Herein, we report the pharmacological effects and targets of ivermectin in prostate cancer. Ivermectin caused G0/G1 cell cycle arrest, induced cell apoptosis and DNA damage, and decreased androgen receptor (AR) signaling in prostate cancer cells. Further in vivo analysis showed ivermectin could suppress 22RV1 xenograft progression. Using integrated omics profiling, including RNA-seq and thermal proteome profiling, the forkhead box protein A1 (FOXA1) and non-homologous end joining (NHEJ) repair executer Ku70/Ku80 were strongly suggested as direct targets of ivermectin in prostate cancer. The interaction of ivermectin and FOXA1 reduced the chromatin accessibility of AR signaling and the G0/G1 cell cycle regulator E2F1, leading to cell proliferation inhibition. The interaction of ivermectin and Ku70/Ku80 impaired the NHEJ repair ability. Cooperating with the downregulation of homologous recombination repair ability after AR signaling inhibition, ivermectin increased intracellular DNA double-strand breaks and finally triggered cell death. Our findings demonstrate the anticancer effect of ivermectin in prostate cancer, indicating that its use may be a new therapeutic approach for prostate cancer.


Assuntos
Fator 3-alfa Nuclear de Hepatócito , Ivermectina , Autoantígeno Ku , Neoplasias da Próstata , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Fator 3-alfa Nuclear de Hepatócito/efeitos dos fármacos , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Ivermectina/farmacologia , Ivermectina/uso terapêutico , Autoantígeno Ku/efeitos dos fármacos , Autoantígeno Ku/metabolismo , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo
16.
Sci Rep ; 12(1): 13373, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927309

RESUMO

Recent studies revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal breaks and recurrent DSB clusters in late-replicating, long neural genes that may give rise to somatic brain mosaicism. The mechanistic factors promoting these different classes of DSBs in neural stem/progenitor cells are not understood. Here, we elucidated the genome-wide landscape of RNA:DNA hybrid structures called "R-loops" in primary neural stem/progenitor cells undergoing aphidicolin-induced, mild replication stress to assess the potential contribution of R-loops to the different, recurrent classes of DNA break "hotspots". We find that R-loops in neural stem/progenitor cells undergoing mild replication stress are present primarily in early-replicating, transcribed regions and in genes with promoter GC skew that are associated with cell lineage-specific processes. Surprisingly, most long, neural genes that form recurrent DSB clusters do not show R-loop formation under conditions of mild replication stress. Our findings are consistent with a role of R-loop-associated processes in promoter-proximal DNA break formation in highly transcribed, early replicating regions but suggest that R-loops do not drive replication stress-induced, recurrent DSB cluster formation in most long, neural genes.


Assuntos
Células-Tronco Neurais , Estruturas R-Loop , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA
17.
DNA Repair (Amst) ; 118: 103386, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35963140

RESUMO

Spermatogenesis is a complex developmental process. During this process, male germ cells from spermatogonia to sperm cells encounter a number of DNA damages. The most severe form of these damages is double-strand breaks (DSBs) deriving from exogenous and endogenous genotoxic insults. DSBs must be correctly repaired in a short time to maintain genomic integrity in the male germ cells. For this purpose, there are four pathways working in repair of DSBs: homologous recombination (HR), classical non-homologous end joining (cNHEJ), alternative end joining (aEJ), and single strand annealing (SSA). While the HR pathway repairs DSBs with a homology-based and error-free manner, the cNHEJ, aEJ, and SSA pathways join free ends in a sequence-independent mechanism. Possible impairments in these DSB repair mechanisms can lead to cell cycle arrest, abnormal meiotic recombination, and ultimately male infertility. In this review, we comprehensively introduce DSB repair pathways being used by male germ cells during spermatogenesis. Also, potential relationship between dysfunction in these pathways and male infertility development are discussed in the light of existing studies.


Assuntos
Quebras de DNA de Cadeia Dupla , Infertilidade Masculina , DNA , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Células Germinativas , Humanos , Infertilidade Masculina/genética , Masculino , Sêmen , Espermatogênese
18.
DNA Repair (Amst) ; 118: 103388, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36037787

RESUMO

Technologies to study DNA double-strand break (DSB) repair have traditionally mostly relied on fluorescence read-outs, either by microscopy or flow cytometry. The advent of high throughput sequencing (HTS) has created fundamentally new opportunities to study the mechanisms underlying DSB repair. Here, we review the suite of HTS-based assays that are used to study three different aspects of DNA repair: detection of broken ends, protein recruitment and pathway usage. We highlight new opportunities that HTS technology offers towards a better understanding of the DSB repair process.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Tecnologia
19.
Nat Struct Mol Biol ; 29(8): 801-812, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35941380

RESUMO

Vertebrate replication forks arrested at interstrand DNA cross-links (ICLs) engage the Fanconi anemia pathway to incise arrested forks, 'unhooking' the ICL and forming a double strand break (DSB) that is repaired by homologous recombination (HR). The FANCP product, SLX4, in complex with the XPF (also known as FANCQ or ERCC4)-ERCC1 endonuclease, mediates ICL unhooking. Whether this mechanism operates at replication fork barriers other than ICLs is unknown. Here, we study the role of mouse SLX4 in HR triggered by a site-specific chromosomal DNA-protein replication fork barrier formed by the Escherichia coli-derived Tus-Ter complex. We show that SLX4-XPF is required for Tus-Ter-induced HR but not for error-free HR induced by a replication-independent DSB. We additionally uncover a role for SLX4-XPF in DSB-induced long-tract gene conversion, an error-prone HR pathway related to break-induced replication. Notably, Slx4 and Xpf mutants that are defective for Tus-Ter-induced HR are hypersensitive to ICLs and also to the DNA-protein cross-linking agents 5-aza-2'-deoxycytidine and zebularine. Collectively, these findings show that SLX4-XPF can process DNA-protein fork barriers for HR and that the Tus-Ter system recapitulates this process.


Assuntos
Anemia de Fanconi , Recombinação Homóloga , Animais , DNA/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Endonucleases/genética , Endonucleases/metabolismo , Anemia de Fanconi/metabolismo , Camundongos
20.
Biochem Soc Trans ; 50(4): 1105-1118, 2022 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-36040211

RESUMO

Failure of cells to process toxic double-strand breaks (DSBs) constitutes a major intrinsic source of genome instability, a hallmark of cancer. In contrast with interphase of the cell cycle, canonical repair pathways in response to DSBs are inactivated in mitosis. Although cell cycle checkpoints prevent transmission of DNA lesions into mitosis under physiological condition, cancer cells frequently display mitotic DNA lesions. In this review, we aim to provide an overview of how mitotic cells process lesions that escape checkpoint surveillance. We outline mechanisms that regulate the mitotic DNA damage response and the different types of lesions that are carried over to mitosis, with a focus on joint DNA molecules arising from under-replication and persistent recombination intermediates, as well as DNA catenanes. Additionally, we discuss the processing pathways that resolve each of these lesions in mitosis. Finally, we address the acute and long-term consequences of unresolved mitotic lesions on cellular fate and genome stability.


Assuntos
Reparo do DNA , Mitose , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Instabilidade Genômica , Humanos
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