RESUMO
In this study we have identified POLθ-S6K-p62 as a novel druggable regulator of radiation response in prostate cancer. Despite significant advances in delivery, radiotherapy continues to negatively affect treatment outcomes and quality of life due to resistance and late toxic effects to the surrounding normal tissues such as bladder and rectum. It is essential to develop new and effective strategies to achieve better control of tumor. We found that ribosomal protein S6K (RPS6KB1) is elevated in human prostate tumors, and contributes to resistance to radiation. As a downstream effector of mTOR signaling, S6K is known to be involved in growth regulation. However, the impact of S6K signaling on radiation response has not been fully explored. Here we show that loss of S6K led to formation of smaller tumors with less metastatic ability in mice. Mechanistically we found that S6K depletion reduced NFκB and SQSTM1 (p62) reporter activity and DNA polymerase θ (POLθ) that is involved in alternate end-joining repair. We further show that the natural compound berberine interacts with S6K in a in a hitherto unreported novel mode and that pharmacological inhibition of S6K with berberine reduces Polθ and downregulates p62 transcriptional activity via NFκB. Loss of S6K or pre-treatment with berberine improved response to radiation in prostate cancer cells and prevented radiation-mediated resurgence of PSA in animals implanted with prostate cancer cells. Notably, silencing POLQ in S6K overexpressing cells enhanced response to radiation suggesting S6K sensitizes prostate cancer cells to radiation via POLQ. Additionally, inhibition of autophagy with CQ potentiated growth inhibition induced by berberine plus radiation. These observations suggest that pharmacological inhibition of S6K with berberine not only downregulates NFκB/p62 signaling to disrupt autophagic flux but also decreases Polθ. Therefore, combination treatment with radiation and berberine inhibits autophagy and alternate end-joining DNA repair, two processes associated with radioresistance leading to increased radiation sensitivity.
Assuntos
NF-kappa B , Neoplasias da Próstata , Tolerância a Radiação , Proteína Sequestossoma-1 , Transdução de Sinais , Masculino , Animais , Humanos , Neoplasias da Próstata/radioterapia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/tratamento farmacológico , Proteína Sequestossoma-1/metabolismo , Proteína Sequestossoma-1/genética , NF-kappa B/metabolismo , NF-kappa B/genética , Transdução de Sinais/efeitos dos fármacos , Camundongos , Tolerância a Radiação/efeitos dos fármacos , Linhagem Celular Tumoral , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genéticaRESUMO
Vision is vital for daily activities, and yet the most common eye diseases-cataracts, DR, ARMD, and glaucoma-lead to blindness in aging eyes. Cataract surgery is one of the most frequently performed surgeries, and the outcome is typically excellent if there is no concomitant pathology present in the visual pathway. In contrast, patients with DR, ARMD and glaucoma often develop significant visual impairment. These often-multifactorial eye problems can have genetic and hereditary components, with recent data supporting the role of DNA damage and repair as significant pathogenic factors. In this article, we discuss the role of DNA damage and the repair deficit in the development of DR, ARMD and glaucoma.
Assuntos
Catarata , Oftalmopatias , Glaucoma , Degeneração Macular , Humanos , Degeneração Macular/complicações , Glaucoma/complicações , Cegueira , Dano ao DNARESUMO
In this work, we developed a DNA dosimeter, consisting of 4-kb DNA strands attached to magnetic streptavidin beads and labeled with fluorescein, to detect double-strand breaks (DSBs). The purpose here was to evaluate whether the DNA dosimeter readings reflect the relative biological effects of 160 kVp and 6 MV X rays. AVarian 600 C/D linac (6 MV) and a Faxitron cabinet X-ray system (160 kVp), both calibrated using traceable methods, were used to deliver high- and low-energy photons, respectively, to DNA dosimeters and multiple cell lines (mNs-5, HT-22 and Daoy). The responses were fit versus dose, and were used to quantify the dose of low-energy photons that produced the same response as that of the high-energy photons, at doses of 3, 6 and 9 Gy. The equivalent doses were utilized to calculate the relative biological effectiveness (RBEDSB and RBEcell survival). Additionally, a neutral comet assay was performed to measure the amount of intracellular DNA DSB, and ultimately the RBEcomet assay. The results of this work showed 160-kVp photon RBE values and 95% confidence intervals of 1.12 ± 0.04 (mNS-5), 1.16 ± 0.06 (HT-22), 1.25 ± 0.09 (Daoy) and 1.21 ± 0.24 (DNA dosimeter) at 9 Gy and 1.32 ± 0.16 (comet assay) at 3 Gy. Within the current error, the DNA dosimeter measured RBEDSB values in agreement with the RBEcell survival and assay from the cell survival and comet assay RBEcomet measurements. These results suggest that the DNA dosimeter can measure the changes in the radiobiological effects from different energy photons.
Assuntos
DNA/genética , Radiometria/instrumentação , Eficiência Biológica Relativa , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Humanos , Raios XRESUMO
Microhomology-mediated end joining (MMEJ) anneals short, imperfect microhomologies flanking DNA breaks, producing repair products with deletions in a Ku- and RAD52-independent fashion. Puzzlingly, MMEJ preferentially selects certain microhomologies over others, even when multiple microhomologies are available. To define rules and parameters for microhomology selection, we altered the length, the position, and the level of mismatches to the microhomologies flanking homothallic switching (HO) endonuclease-induced breaks and assessed their effect on MMEJ frequency and the types of repair product formation. We found that microhomology of eight to 20 base pairs carrying no more than 20% mismatches efficiently induced MMEJ. Deletion of MSH6 did not impact MMEJ frequency. MMEJ preferentially chose a microhomology pair that was more proximal from the break. Interestingly, MMEJ events preferentially retained the centromere proximal side of the HO break, while the sequences proximal to the telomere were frequently deleted. The asymmetry in the deletional profile among MMEJ products was reduced when HO was induced on the circular chromosome. The results provide insight into how cells search and select microhomologies for MMEJ in budding yeast.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência/genéticaRESUMO
Ribonucleoside monophosphates (rNMPs) mis-incorporated during DNA replication are removed by RNase H2-dependent excision repair or by topoisomerase I (Top1)-catalyzed cleavage. The cleavage of rNMPs by Top1 produces 3' ends harboring terminal adducts, such as 2',3'-cyclic phosphate or Top1 cleavage complex (Top1cc), and leads to frequent mutagenesis and DNA damage checkpoint induction. We surveyed a range of candidate enzymes from Saccharomyces cerevisiae for potential roles in Top1-dependent genomic rNMP removal. Genetic and biochemical analyses reveal that Apn2 resolves phosphotyrosine-DNA conjugates, terminal 2',3'-cyclic phosphates, and their hydrolyzed products. APN2 also suppresses 2-base pair (bp) slippage mutagenesis in RNH201-deficient cells. Our results define additional activities of Apn2 in resolving a wide range of 3' end blocks and identify a role for Apn2 in maintaining genome integrity during rNMP repair.
Assuntos
Pareamento de Bases/genética , Reparo do DNA/genética , Replicação do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Ribonucleotídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Regiões 3' não Traduzidas/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Fúngico/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Genoma Fúngico/genética , Mutagênese/genética , Ribonucleases/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.
RESUMO
Emerging evidence indicates that adipose stromal cells (ASC) are recruited to enhance cancer development. In this study, we examined the role these adipocyte progenitors play relating to intercellular communication in obesity-associated endometrial cancer. This is particularly relevant given that gap junctions have been implicated in tumor suppression. Examining the effects of ASCs on the transcriptome of endometrial epithelial cells (EEC) in an in vitro coculture system revealed transcriptional repression of GJA1 (encoding the gap junction protein Cx43) and other genes related to intercellular communication. This repression was recapitulated in an obesity mouse model of endometrial cancer. Furthermore, inhibition of plasminogen activator inhibitor 1 (PAI-1), which was the most abundant ASC adipokine, led to reversal of cellular distribution associated with the GJA1 repression profile, suggesting that PAI-1 may mediate actions of ASC on transcriptional regulation in EEC. In an endometrial cancer cohort (n = 141), DNA hypermethylation of GJA1 and related loci TJP2 and PRKCA was observed in primary endometrial endometrioid tumors and was associated with obesity. Pharmacologic reversal of DNA methylation enhanced gap-junction intercellular communication and cell-cell interactions in vitro. Restoring Cx43 expression in endometrial cancer cells reduced cellular migration; conversely, depletion of Cx43 increased cell migration in immortalized normal EEC. Our data suggest that persistent repression by ASC adipokines leads to promoter hypermethylation of GJA1 and related genes in the endometrium, triggering long-term silencing of these loci in endometrial tumors of obese patients. SIGNIFICANCE: Studies reveal that adipose-derived stem cells in endometrial cancer pathogenesis influence epigenetic repression of gap junction loci, which suggests targeting of gap junction activity as a preventive strategy for obesity-associated endometrial cancer.
Assuntos
Adipocinas/farmacologia , Tecido Adiposo/patologia , Comunicação Celular , Conexina 43/genética , Neoplasias do Endométrio/patologia , Repressão Epigenética , Obesidade/complicações , Tecido Adiposo/metabolismo , Animais , Movimento Celular , Células Cultivadas , Conexina 43/metabolismo , Dieta Hiperlipídica/efeitos adversos , Neoplasias do Endométrio/etiologia , Neoplasias do Endométrio/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Junções Comunicantes , Humanos , Masculino , Camundongos , Camundongos Knockout , Obesidade/fisiopatologia , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic resection of the DNA break ends. The current model, being based primarily on genetic analyses in Saccharomyces cerevisiae and companion biochemical reconstitution studies, posits that end resection proceeds in two distinct stages. Specifically, the initiation of resection is mediated by the nuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex in conjunction with its cofactor Sae2, and long-range resection is carried out by exonuclease 1 (Exo1) or the Sgs1-Top3-Rmi1-Dna2 ensemble. Using fully reconstituted systems, we show here that DNA with ends occluded by the DNA end-joining factor Ku70-Ku80 becomes a suitable substrate for long-range 5'-3' resection when a nick is introduced at a locale proximal to one of the Ku-bound DNA ends. We also show that Sgs1 can unwind duplex DNA harboring a nick, in a manner dependent on a species-specific interaction with the ssDNA-binding factor replication protein A (RPA). These biochemical systems and results will be valuable for guiding future endeavors directed at delineating the mechanistic intricacy of DNA end resection in eukaryotes.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , RecQ Helicases/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , DNA Helicases/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/genética , Recombinação Homóloga , RecQ Helicases/genética , Proteína de Replicação A/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Yeast Rad1-Rad10 (XPF-ERCC1 in mammals) incises UV, oxidation, and cross-linking agent-induced DNA lesions, and contributes to multiple DNA repair pathways. To determine how Rad1-Rad10 catalyzes inter-strand crosslink repair (ICLR), we examined sensitivity to ICLs from yeast deleted for SAW1 and SLX4, which encode proteins that interact physically with Rad1-Rad10 and bind stalled replication forks. Saw1, Slx1, and Slx4 are critical for replication-coupled ICLR in mus81 deficient cells. Two rad1 mutations that disrupt interactions between Rpa1 and Rad1-Rad10 selectively disable non-nucleotide excision repair (NER) function, but retain UV lesion repair. Mutations in the analogous region of XPF also compromised XPF interactions with Rpa1 and Slx4, and are proficient in NER but deficient in ICLR and direct repeat recombination. We propose that Rad1-Rad10 makes distinct contributions to ICLR depending on cell cycle phase: in G1, Rad1-Rad10 removes ICL via NER, whereas in S/G2, Rad1-Rad10 facilitates NER-independent replication-coupled ICLR.
Assuntos
Dano ao DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Animais , Células CHO , Ciclo Celular/genética , Cricetulus , Reagentes de Ligações Cruzadas/toxicidade , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Microscopia Intravital , Mutagênese Sítio-Dirigida , Mutação , Proteínas de Saccharomyces cerevisiae/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética , Raios Ultravioleta/efeitos adversosRESUMO
PURPOSE: Many types of dosimeters are used to measure radiation dose and calibrate radiotherapy equipment, but none directly measure the biological effect of this dose. The purpose here is to create a dosimeter that can measure the probability of double-strand breaks (DSB) for DNA, which is directly related to the biological effect of radiation. METHODS: A DNA dosimeter, consisting of magnetic streptavidin beads attached to four kilobase pair DNA strands labeled with biotin and fluorescein amidite (FAM) on opposing ends, was suspended in phosphate-buffered saline (PBS). Fifty microliter samples were placed in plastic tubes inside a water tank setup and irradiated at the dose levels of 25, 50, 100, 150, and 200 Gy. After irradiation, the dosimeters were mechanically separated into beads (intact DNA) and supernatant (broken DNA/FAM) using a magnet. The fluorescence was read and the probability of DSB was calculated. This DNA dosimeter response was benchmarked against a Southern blot analysis technique for the measurement of DSB probability. RESULTS: For the DNA dosimeter, the probabilities of DSB at the dose levels of 25, 50, 100, 150, and 200 Gy were 0.043, 0.081, 0.149, 0.196, and 0.242, respectively, and the standard errors of the mean were 0.002, 0.003, 0.006, 0.005, and 0.011, respectively. For the Southern blot method, the probabilities of DSB at the dose levels of 25, 50, 100, 150, and 200 Gy were 0.053, 0.105, 0.198, 0.235, and 0.264, respectively, and the standard errors of the mean were 0.013, 0.024, 0.040, 0.044, and 0.063, respectively. CONCLUSIONS: A DNA dosimeter can accurately determine the probability of DNA double-strand break (DSB), one of the most toxic effects of radiotherapy, for absorbed radiation doses from 25 to 200 Gy. This is an important step in demonstrating the viability of DNA dosimeters as a measurement technique for radiation.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , Radiometria/métodos , Dosagem Radioterapêutica , Animais , Biotina , Southern Blotting , Desenho de Equipamento , Humanos , Probabilidade , Dosímetros de Radiação , Radiometria/instrumentação , Cloreto de Sódio , Estreptavidina , ÁguaRESUMO
DNA double-strand breaks (DSBs) are induced by a variety of genotoxic agents, including ionizing radiation and chemotherapy drugs for treating cancers. The elimination of DSBs proceeds via distinctive error-free and error-prone pathways. Repair by homologous recombination (HR) is largely error-free and mediated by RAD51/BRCA2 gene products. Classical non-homologous end joining (C-NHEJ) requires the Ku heterodimer and can efficiently rejoin breaks, with occasional loss or gain of DNA information. Recently, evidence has unveiled another DNA end-joining mechanism that is independent of recombination factors and Ku proteins, termed alternative non-homologous end joining (A-NHEJ). While A-NHEJ-mediated repair does not require homology, in a subtype of A-NHEJ, DSB breaks are sealed by microhomology (MH)-mediated base-pairing of DNA single strands, followed by nucleolytic trimming of DNA flaps, DNA gap filling, and DNA ligation, yielding products that are always associated with DNA deletion. This highly error-prone DSB repair pathway is termed microhomology-mediated end joining (MMEJ). Dissecting the mechanisms of MMEJ is of great interest because of its potential to destabilize the genome through gene deletions and chromosomal rearrangements in cells deficient in canonical repair pathways, including HR and C-NHEJ. In addition, evidence now suggests that MMEJ plays a physiological role in normal cells.
Assuntos
Proteína BRCA2/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Autoantígeno Ku/metabolismo , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Animais , Proteína BRCA2/genética , Aberrações Cromossômicas , Deleção de Genes , Humanos , Autoantígeno Ku/genética , Rad51 Recombinase/genéticaRESUMO
Microhomology (MH) flanking a DNA double-strand break (DSB) drives chromosomal rearrangements but its role in mutagenesis has not yet been analyzed. Here we determined the mutation frequency of a URA3 reporter gene placed at multiple locations distal to a DSB, which is flanked by different sizes (15-, 18-, or 203-bp) of direct repeat sequences for efficient repair in budding yeast. Induction of a DSB accumulates mutations in the reporter gene situated up to 14-kb distal to the 15-bp MH, but more modestly to those carrying 18- and 203-bp or no homology. Increased mutagenesis in MH-mediated end joining (MMEJ) appears coupled to its slower repair kinetics and the extensive resection occurring at flanking DNA. Chromosomal translocations via MMEJ also elevate mutagenesis of the flanking DNA sequences 7.1 kb distal to the breakpoint junction as compared to those without MH. The results suggest that MMEJ could destabilize genomes by triggering structural alterations and increasing mutation burden.
Assuntos
Reparo do DNA por Junção de Extremidades/genética , DNA/genética , Mutagênese/genética , Proteínas de Saccharomyces cerevisiae/genética , Cromossomos/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Galactose/genética , Cinética , Mutagênese/efeitos dos fármacos , Mutagênese Insercional , Saccharomyces cerevisiae , Translocação Genética/efeitos dos fármacos , Translocação Genética/genéticaRESUMO
Prevalence of microhomology (MH) at the breakpoint junctions in somatic and germ-line chromosomal rearrangements and in the programmed immune receptor rearrangements from cells deficient in classical end joining reveals an enigmatic process called MH-mediated end joining (MMEJ). MMEJ repairs DNA double strand breaks (DSBs) by annealing flanking MH and deleting genetic information at the repair junctions from yeast to humans. Being genetically distinct from canonical DNA DSB pathways, MMEJ is involved with the fusions of eroded/uncapped telomeres as well as with the assembly of chromosome fragments in chromothripsis. In this review article, we will discuss an up-to-date model representing the MMEJ process and the mechanism by which cells regulate MMEJ to limit repair-associated mutagenesis. We will also describe the possible therapeutic gains resulting from the inhibition of MMEJ in recombination deficient cancers. Lastly, we will embark on two contentious issues associated with MMEJ such as the significance of MH at the repair junction to be the hallmark of MMEJ and the relationship of MMEJ to other mechanistically related DSB repair pathways.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Modelos Genéticos , Animais , Ciclo Celular/genética , Sobrevivência Celular/genética , Recombinação Homóloga , Humanos , Mutação INDELRESUMO
Break-induced replication (BIR) has been implicated in restoring eroded telomeres and collapsed replication forks via single-ended invasion and extensive DNA synthesis on the recipient chromosome. Unlike other recombination subtypes, DNA synthesis in BIR likely relies heavily on mechanisms enabling efficient fork progression such as chromatin modification. Herein we report that deletion of HST3 and HST4, two redundant de-acetylases of histone H3 Lysine 56 (H3K56), inhibits BIR, sensitizes checkpoint deficient cells to deoxyribonucleotide triphosphate pool depletion, and elevates translocation-type gross chromosomal rearrangements (GCR). The basis for deficiency in BIR and gene conversion with long gap synthesis in hst3Δ hst4Δ cells can be traced to a defect in extensive DNA synthesis. Distinct from other cellular defects associated with deletion of HST3 and HST4 including thermo-sensitivity and elevated spontaneous mutagenesis, the BIR defect in hst3Δ hst4Δ cannot be offset by the deletion of RAD17 or MMS22, but rather by the loss of RTT109 or ASF1, or in combination with the H3K56R mutation, which also restores tolerance to replication stress in mrc1 mutants. Our studies suggest that acetylation of H3K56 limits extensive repair synthesis and interferes with efficient fork progression in BIR.
Assuntos
Replicação do DNA/genética , Histona Desacetilases/genética , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Acetilação , Cromatina/genética , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA/genética , Histonas/genética , Humanos , Mutação , Saccharomyces cerevisiae/genética , Telômero/genéticaRESUMO
Chromosomal structural change triggers carcinogenesis and the formation of other genetic diseases. The breakpoint junctions of these rearrangements often contain small overlapping sequences called "microhomology," yet the genetic pathway(s) responsible have yet to be defined. We report a simple genetic system to detect microhomology-mediated repair (MHMR) events after a DNA double-strand break (DSB) in budding yeast cells. MHMR using >15 bp operates as a single-strand annealing variant, requiring the non-essential DNA polymerase subunit Pol32. MHMR is inhibited by sequence mismatches, but independent of extensive DNA synthesis like break-induced replication. However, MHMR using less than 14 bp is genetically distinct from that using longer microhomology and far less efficient for the repair of distant DSBs. MHMR catalyzes chromosomal translocation almost as efficiently as intra-chromosomal repair. The results suggest that the intrinsic annealing propensity between microhomology sequences efficiently leads to chromosomal rearrangements.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Replicação do DNA/genética , Translocação Genética/genética , Aberrações Cromossômicas , Cromossomos/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Recombinação Genética , Saccharomyces cerevisiaeRESUMO
DNA recombination pathways are regulated by the cell cycle to coordinate with replication. Cyclin-dependent kinase (Cdk1) promotes efficient 5' strand resection at DNA double-strand breaks (DSBs), the initial step of homologous recombination and damage checkpoint activation. The Mre11-Rad50-Xrs2 complex with Sae2 initiates resection, whereas two nucleases, Exo1 and Dna2, and the DNA helicase-topoisomerase complex Sgs1-Top3-Rmi1 generate longer ssDNA at DSBs. Using Saccharomyces cerevisiae, we provide evidence for Cdk1-dependent phosphorylation of the resection nuclease Dna2 at Thr4, Ser17 and Ser237 that stimulates its recruitment to DSBs, resection and subsequent Mec1-dependent phosphorylation. Poorly recruited dna2T4A S17A S237A and dna2ΔN248 mutant proteins promote resection only in the presence of Exo1, suggesting cross-talk between Dna2- and Exo1-dependent resection pathways.
Assuntos
Proteína Quinase CDC2/fisiologia , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Proteína Quinase CDC2/química , Exodesoxirribonucleases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Genéticos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , CoesinasRESUMO
Single-stranded DNA constitutes an important early intermediate for homologous recombination and damage-induced cell cycle checkpoint activation. In Saccharomyces cerevisiae, efficient double-strand break (DSB) end resection requires several enzymes; Mre11/Rad50/Xrs2 (MRX) and Sae2 are implicated in the onset of 5'-strand resection, whereas Sgs1/Top3/Rmi1 with Dna2 and Exo1 are involved in extensive resection. However, the molecular events leading to a switch from the MRX/Sae2-dependent initiation to the Exo1- and Dna2-dependent resection remain unclear. Here, we show that MRX recruits Dna2 nuclease to DSB ends. MRX also stimulates recruitment of Exo1 and antagonizes excess binding of the Ku complex to DSB ends. Using resection assay with purified enzymes in vitro, we found that Ku and MRX regulate the nuclease activity of Exo1 in an opposite way. Efficient loading of Dna2 and Exo1 requires neither Sae2 nor Mre11 nuclease activities. However, Mre11 nuclease activity is essential for resection in the absence of extensive resection enzymes. The results provide new insights into how MRX catalyses end resection and recombination initiation.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imunoprecipitação da Cromatina , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Saccharomyces cerevisiaeRESUMO
Cell cycle plays a crucial role in regulating the pathway used to repair DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, homologous recombination is primarily limited to non-G(1) cells as the formation of recombinogenic single-stranded DNA requires CDK1-dependent 5' to 3' resection of DNA ends. However, the effect of cell cycle on non-homologous end joining (NHEJ) is not yet clearly defined. Using an assay to quantitatively measure the contributions of each repair pathway to repair product formation and cellular survival after DSB induction, we found that NHEJ is most efficient at G(1), and markedly repressed at G(2). Repression of NHEJ at G(2) is achieved by efficient end resection and by the reduced association of core NHEJ proteins with DNA breaks, both of which depend on the CDK1 activity. Importantly, repression of 5' end resection by CDK1 inhibition at G(2) alone did not fully restore either physical association of Ku/Dnl4-Lif1 with DSBs or NHEJ proficiency to the level at G(1). Expression of excess Ku can partially offset the inhibition of end joining at G(2). The results suggest that regulation of Ku/Dnl4-Lif1 affinity for DNA ends may contribute to the cell cycle-dependent modulation of NHEJ efficiency.
Assuntos
Proteína Quinase CDC2/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Ciclo Celular , Sobrevivência Celular , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , Enzimas Reparadoras do DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remain unknown. We monitored 5'-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5'-strand resection. We show that the Mre11-Rad50-Xrs2 complex (MRX) initiates 5' degradation, whereas Sgs1 and Dna2 degrade 5' strands exposing long 3' strands. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In exo1Deltasgs1Delta double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.