RESUMO
Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that â¼21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells. We refer to these as diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. In Arabidopsis, diRNAs are recruited by Argonaute 2 (AGO2) to mediate DSB repair. Knock down of Dicer or Ago2 in human cells reduces DSB repair. Our findings reveal a conserved function for small RNAs in the DSB repair pathway. We propose that diRNAs may function as guide molecules directing chromatin modifications or the recruitment of protein complexes to DSB sites to facilitate repair.
Assuntos
Arabidopsis/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , RNA de Plantas/metabolismo , RNA não Traduzido/metabolismo , RNA Helicases DEAD-box/metabolismo , Humanos , Ribonuclease III/metabolismoRESUMO
We report here the successful labelling of meiotic prophase I DNA synthesis in the flowering plant, Arabidopsis thaliana. Incorporation of the thymidine analogue, EdU, enables visualisation of the footprints of recombinational repair of programmed meiotic DNA double-strand breaks (DSB), with ~400 discrete, SPO11-dependent, EdU-labelled chromosomal foci clearly visible at pachytene and later stages of meiosis. This number equates well with previous estimations of 200-300 DNA double-strand breaks per meiosis in Arabidopsis, confirming the power of this approach to detect the repair of most or all SPO11-dependent meiotic DSB repair events. The chromosomal distribution of these DNA-synthesis foci accords with that of early recombination markers and MLH1, which marks Class I crossover sites. Approximately 10 inter-homologue cross-overs (CO) have been shown to occur in each Arabidopsis male meiosis and, athough very probably under-estimated, an equivalent number of inter-homologue gene conversions (GC) have been described. Thus, at least 90% of meiotic recombination events, and very probably more, have not previously been accessible for analysis. Visual examination of the patterns of the foci on the synapsed pachytene chromosomes corresponds well with expectations from the different mechanisms of meiotic recombination and notably, no evidence for long Break-Induced Replication DNA synthesis tracts was found. Labelling of meiotic prophase I, SPO11-dependent DNA synthesis holds great promise for further understanding of the molecular mechanisms of meiotic recombination, at the heart of reproduction and evolution of eukaryotes.
Assuntos
Arabidopsis , Quebras de DNA de Cadeia Dupla , Meiose , Arabidopsis/genética , Meiose/genética , Reparo do DNA/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromossomos de Plantas/genética , Prófase Meiótica I/genética , Troca Genética , Replicação do DNA/genéticaRESUMO
Ensuring balanced distribution of chromosomes in gametes, meiotic recombination is essential for fertility in most sexually reproducing organisms. The repair of the programmed DNA double strand breaks that initiate meiotic recombination requires two DNA strand-exchange proteins, RAD51 and DMC1, to search for and invade an intact DNA molecule on the homologous chromosome. DMC1 is meiosis-specific, while RAD51 is essential for both mitotic and meiotic homologous recombination. DMC1 is the main catalytically active strand-exchange protein during meiosis, while this activity of RAD51 is downregulated. RAD51 is however an essential cofactor in meiosis, supporting the function of DMC1. This work presents a study of the mechanism(s) involved in this and our results point to DMC1 being, at least, a major actor in the meiotic suppression of the RAD51 strand-exchange activity in plants. Ectopic expression of DMC1 in somatic cells renders plants hypersensitive to DNA damage and specifically impairs RAD51-dependent homologous recombination. DNA damage-induced RAD51 focus formation in somatic cells is not however suppressed by ectopic expression of DMC1. Interestingly, DMC1 also forms damage-induced foci in these cells and we further show that the ability of DMC1 to prevent RAD51-mediated recombination is associated with local assembly of DMC1 at DNA breaks. In support of our hypothesis, expression of a dominant negative DMC1 protein in meiosis impairs RAD51-mediated DSB repair. We propose that DMC1 acts to prevent RAD51-mediated recombination in Arabidopsis and that this down-regulation requires local assembly of DMC1 nucleofilaments.
Assuntos
Arabidopsis , Proteínas de Saccharomyces cerevisiae , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Recombinação Homóloga/genética , Meiose/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Throughout their lifecycle, plants are subjected to DNA damage from various sources, both environmental and endogenous. Investigating the mechanisms of the DNA damage response (DDR) is essential to unravel how plants adapt to the changing environment, which can induce varying amounts of DNA damage. Using a combination of whole-mount single-molecule RNA fluorescence in situ hybridization (WM-smFISH) and plant cell cycle reporter lines, we investigated the transcriptional activation of a key homologous recombination (HR) gene, RAD51, in response to increasing amounts of DNA damage in Arabidopsis thaliana roots. The results uncover consistent variations in RAD51 transcriptional response and cell cycle arrest among distinct cell types and developmental zones. Furthermore, we demonstrate that DNA damage induced by genotoxic stress results in RAD51 transcription throughout the whole cell cycle, dissociating its traditional link with S/G2 phases. This work advances the current comprehension of DNA damage response in plants by demonstrating quantitative differences in DDR activation. In addition, it reveals new associations with the cell cycle and cell types, providing crucial insights for further studies of the broader response mechanisms in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ciclo Celular , Dano ao DNA , Regulação da Expressão Gênica de Plantas , Raízes de Plantas , Rad51 Recombinase , Transcrição Gênica , Arabidopsis/genética , Raízes de Plantas/genética , Raízes de Plantas/citologia , Ciclo Celular/genética , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMO
An essential component of the homologous recombination machinery in eukaryotes, the RAD54 protein is a member of the SWI2/SNF2 family of helicases with dsDNA-dependent ATPase, DNA translocase, DNA supercoiling and chromatin remodelling activities. It is a motor protein that translocates along dsDNA and performs multiple functions in homologous recombination. In particular, RAD54 is an essential cofactor for regulating RAD51 activity. It stabilizes the RAD51 nucleofilament, remodels nucleosomes, and stimulates the homology search and strand invasion activities of RAD51. Accordingly, deletion of RAD54 has dramatic consequences on DNA damage repair in mitotic cells. In contrast, its role in meiotic recombination is less clear. RAD54 is essential for meiotic recombination in Drosophila and C. elegans, but plays minor roles in yeast and mammals. We present here characterization of the roles of RAD54 in meiotic recombination in the model plant Arabidopsis thaliana. Absence of RAD54 has no detectable effect on meiotic recombination in otherwise wild-type plants but RAD54 becomes essential for meiotic DSB repair in absence of DMC1. In Arabidopsis, dmc1 mutants have an achiasmate meiosis, in which RAD51 repairs meiotic DSBs. Lack of RAD54 leads to meiotic chromosomal fragmentation in absence of DMC1. The action of RAD54 in meiotic RAD51 activity is thus mainly downstream of the role of RAD51 in supporting the activity of DMC1. Equivalent analyses show no effect on meiosis of combining dmc1 with the mutants of the RAD51-mediators RAD51B, RAD51D and XRCC2. RAD54 is thus required for repair of meiotic DSBs by RAD51 and the absence of meiotic phenotype in rad54 plants is a consequence of RAD51 playing a RAD54-independent supporting role to DMC1 in meiotic recombination.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Meiose , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/deficiência , DNA Helicases/genética , Proteínas de Ligação a DNA , Genes Essenciais , Meiose/genética , Mutação , Rad51 Recombinase/genética , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas RepressorasRESUMO
Production of gametes of halved ploidy for sexual reproduction requires a specialized cell division called meiosis. The fusion of two gametes restores the original ploidy in the new generation, and meiosis thus stabilizes ploidy across generations. To ensure balanced distribution of chromosomes, pairs of homologous chromosomes (homologs) must recognize each other and pair in the first meiotic division. Recombination plays a key role in this in most studied species, but it is not the only actor and particular chromosomal regions are known to facilitate the meiotic pairing of homologs. In this review, we focus on the roles of centromeres and in particular on the clustering and pairwise associations of nonhomologous centromeres that precede stable pairing between homologs. Although details vary from species to species, it is becoming increasingly clear that these associations play active roles in the meiotic chromosome pairing process, analogous to those of the telomere bouquet.
Assuntos
Centrômero/metabolismo , Pareamento Cromossômico/fisiologia , Animais , Centrômero/genética , Drosophila/genética , Heterocromatina/fisiologia , Meiose/fisiologia , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Telômero/metabolismoRESUMO
DNA entanglements and supercoiling arise frequently during normal DNA metabolism. DNA topoisomerases are highly conserved enzymes that resolve the topological problems that these structures create. Topoisomerase II (TOPII) releases topological stress in DNA by removing DNA supercoils through breaking the two DNA strands, passing a DNA duplex through the break and religating the broken strands. TOPII performs key DNA metabolic roles essential for DNA replication, chromosome condensation, heterochromatin metabolism, telomere disentanglement, centromere decatenation, transmission of crossover (CO) interference, interlock resolution and chromosome segregation in several model organisms. In this study, we reveal the endogenous role of Arabidopsis thaliana TOPII in normal root growth and cell cycle, and mitotic DNA repair via homologous recombination. Additionally, we show that the protein is required for meiotic DSB repair progression, but not for CO formation. We propose that TOPII might promote mitotic HR DNA repair by relieving stress needed for HR strand invasion and D-loop formation.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Reparo do DNA/fisiologia , DNA Topoisomerases Tipo II/genética , Recombinação Homóloga , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/metabolismo , Segregação de Cromossomos , Cromossomos de Plantas , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA Topoisomerases Tipo II/metabolismo , Raios gama , Meiose , Mitomicina/farmacologia , MutaçãoRESUMO
Homologous recombination is a key process for maintaining genome integrity and diversity. In eukaryotes, the nucleosome structure of chromatin inhibits the progression of homologous recombination. The DNA repair and recombination protein RAD54 alters the chromatin structure via nucleosome sliding to enable homology searches. For homologous recombination to progress, appropriate recruitment and dissociation of RAD54 is required at the site of homologous recombination; however, little is known about the mechanism regulating RAD54 dynamics in chromatin. Here, we reveal that the histone demethylase LYSINE-SPECIFIC DEMETHYLASE1-LIKE 1 (LDL1) regulates the dissociation of RAD54 at damaged sites during homologous recombination repair in the somatic cells of Arabidopsis (Arabidopsis thaliana). Depletion of LDL1 leads to an overaccumulation of RAD54 at damaged sites with DNA double-strand breaks. Moreover, RAD54 accumulates at damaged sites by recognizing histone H3 Lys 4 di-methylation (H3K4me2); the frequency of the interaction between RAD54 and H3K4me2 increased in the ldl1 mutant with DNA double-strand breaks. We propose that LDL1 removes RAD54 at damaged sites by demethylating H3K4me2 during homologous recombination repair and thereby maintains genome stability in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , DNA Helicases/metabolismo , Histona Desmetilases/metabolismo , Reparo de DNA por Recombinação , Arabidopsis/genética , Histonas/metabolismoRESUMO
Replicative erosion of telomeres is naturally compensated by telomerase and studies in yeast and vertebrates show that homologous recombination can compensate for the absence of telomerase. We show that RAD51 protein, which catalyzes the key strand-invasion step of homologous recombination, is localized at Arabidopsis telomeres in absence of telomerase. Blocking the strand-transfer activity of the RAD51 in telomerase mutant plants results in a strikingly earlier onset of developmental defects, accompanied by increased numbers of end-to-end chromosome fusions. Imposing replication stress through knockout of RNaseH2 increases numbers of chromosome fusions and reduces the survival of these plants deficient for telomerase and homologous recombination. This finding suggests that RAD51-dependent homologous recombination acts as an essential backup to the telomerase for compensation of replicative telomere loss to ensure genome stability. Furthermore, we show that this positive role of RAD51 in telomere stability is dependent on the RTEL1 helicase. We propose that a RAD51 dependent break-induced replication process is activated in cells lacking telomerase activity, with RTEL1 responsible for D-loop dissolution after telomere replication.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , DNA Helicases/fisiologia , Rad51 Recombinase/fisiologia , Encurtamento do Telômero , Arabidopsis/enzimologia , Proteínas de Arabidopsis/análise , Replicação do DNA , Instabilidade Genômica , Recombinação Homóloga , Mutação , Rad51 Recombinase/análise , Sequências Repetitivas de Ácido Nucleico , Ribonucleases/genética , Processos Estocásticos , Telomerase/genética , Telômero/químicaRESUMO
Despite intensive searches, few proteins involved in telomere homeostasis have been identified in plants. Here, we used pull-down assays to identify potential telomeric interactors in the model plant species Arabidopsis (Arabidopsis thaliana). We identified the candidate protein GH1-HMGA1 (also known as HON4), an uncharacterized linker histone protein of the High Mobility Group Protein A (HMGA) family in plants. HMGAs are architectural transcription factors and have been suggested to function in DNA damage repair, but their precise biological roles remain unclear. Here, we show that GH1-HMGA1 is required for efficient DNA damage repair and telomere integrity in Arabidopsis. GH1-HMGA1 mutants exhibit developmental and growth defects, accompanied by ploidy defects, increased telomere dysfunction-induced foci, mitotic anaphase bridges, and degraded telomeres. Furthermore, mutants have a higher sensitivity to genotoxic agents such as mitomycin C and γ-irradiation. Our work also suggests that GH1-HMGA1 is involved directly in the repair process by allowing the completion of homologous recombination.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas HMGA/metabolismo , Histonas/metabolismo , Telômero/metabolismo , Arabidopsis/crescimento & desenvolvimento , Cromatina/metabolismo , DNA Bacteriano/genética , Fluorescência , Recombinação Homóloga/genética , Mutação/genética , Ligação Proteica , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Structure-specific endonucleases act to repair potentially toxic structures produced by recombination and DNA replication, ensuring proper segregation of the genetic material to daughter cells during mitosis and meiosis. Arabidopsis thaliana has two putative homologs of the resolvase (structure-specific endonuclease): GEN1/Yen1. Knockout of resolvase genes GEN1 and SEND1, individually or together, has no detectable effect on growth, fertility, or sensitivity to DNA damage. However, combined absence of the endonucleases MUS81 and SEND1 results in severe developmental defects, spontaneous cell death, and genome instability. A similar effect is not seen in mus81 gen1 plants, which develop normally and are fertile. Absence of RAD51 does not rescue mus81 send1, pointing to roles of these proteins in DNA replication rather than DNA break repair. The enrichment of S-phase histone γ-H2AX foci and a striking loss of telomeric DNA in mus81 send1 further support this interpretation. SEND1 has at most a minor role in resolution of the Holliday junction but acts as an essential backup to MUS81 for resolution of toxic replication structures to ensure genome stability and to maintain telomere integrity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endonucleases/metabolismo , Resolvases de Junção Holliday/metabolismo , Telômero/metabolismo , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Ciclo Celular , Cromossomos de Plantas/genética , Reparo do DNA , Replicação do DNA , DNA Bacteriano/genética , Instabilidade Genômica , Meiose , Mutagênese Insercional/genética , Mutação/genética , Fenótipo , Rad51 Recombinase/metabolismoRESUMO
Plants have various defense mechanisms against environmental stresses that induce DNA damage. Genetic and biochemical analyses have revealed the sensing and signaling of DNA damage, but little is known about subnuclear dynamics in response to DNA damage in living plant cells. Here, we observed that the chromatin remodeling factor RAD54, which is involved in DNA repair via the homologous recombination pathway, formed subnuclear foci (termed RAD54 foci) in Arabidopsis thaliana after induction of DNA double-strand breaks. The appearance of RAD54 foci was dependent on the ATAXIA-TELANGIECTASIA MUTATED-SUPPRESSOR OF GAMMA RESPONSE 1 pathway, and RAD54 foci were co-localized with γH2AX signals. Laser irradiation of a subnuclear area demonstrated that in living cells RAD54 was specifically accumulated at the damaged site. In addition, the formation of RAD54 foci showed specificity for cell type and region. We conclude that RAD54 foci correspond to DNA repair foci in A. thaliana.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Reparo do DNA/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA/genética , DNA Helicases/genética , Reparo do DNA/genéticaRESUMO
The WEE1 kinase is an essential cell cycle checkpoint regulator in Arabidopsis thaliana plants experiencing replication defects. Whereas under non-stress conditions WEE1-deficient plants develop normally, they fail to adapt to replication inhibitory conditions, resulting in the accumulation of DNA damage and loss of cell division competence. We identified mutant alleles of the genes encoding subunits of the ribonuclease H2 (RNase H2) complex, known for its role in removing ribonucleotides from DNA-RNA duplexes, as suppressor mutants of WEE1 knockout plants. RNase H2 deficiency triggered an increase in homologous recombination (HR), correlated with the accumulation of γ-H2AX foci. However, as HR negatively impacts the growth of WEE1-deficient plants under replication stress, it cannot account for the rescue of the replication defects of the WEE1 knockout plants. Rather, the observed increase in ribonucleotide incorporation in DNA indicates that the substitution of deoxynucleotide with ribonucleotide abolishes the need for WEE1 under replication stress. Strikingly, increased ribonucleotide incorporation in DNA correlated with the occurrence of small base pair deletions, identifying the RNase H2 complex as an important suppressor of genome instability.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Pontos de Checagem do Ciclo Celular , Instabilidade Genômica , Proteínas Serina-Treonina Quinases/metabolismo , Ribonuclease H/deficiência , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Pareamento de Bases , Sequência de Bases , Domínio Catalítico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Genes de Plantas , Instabilidade Genômica/efeitos dos fármacos , Hidroxiureia/farmacologia , Dados de Sequência Molecular , Mutação/genética , Taxa de Mutação , Recombinação Genética/genética , Ribonuclease H/química , Ribonuclease H/genética , Ribonuclease H/metabolismo , Ribonucleotídeos/metabolismoRESUMO
The telomeres of linear eukaryotic chromosomes are protected by caps consisting of evolutionarily conserved nucleoprotein complexes. Telomere dysfunction leads to recombination of chromosome ends and this can result in fusions which initiate chromosomal breakage-fusion-bridge cycles, causing genomic instability and potentially cell death or cancer. We hypothesize that in the absence of the recombination pathways implicated in these fusions, deprotected chromosome ends will instead be eroded by nucleases, also leading to the loss of genes and cell death. In this work, we set out to specifically test this hypothesis in the plant, Arabidopsis. Telomere protection in Arabidopsis implicates KU and CST and their absence leads to chromosome fusions, severe genomic instability and dramatic developmental defects. We have analysed the involvement of end-joining recombination pathways in telomere fusions and the consequences of this on genomic instability and growth. Strikingly, the absence of the multiple end-joining pathways eliminates chromosome fusion and restores normal growth and development to cst ku80 mutant plants. It is thus the chromosomal fusions, per se, which are the underlying cause of the severe developmental defects. This rescue is mediated by telomerase-dependent telomere extension, revealing a competition between telomerase and end-joining recombination proteins for access to deprotected telomeres.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Reparo do DNA por Junção de Extremidades , Recombinação Genética , Telomerase/fisiologia , Homeostase do Telômero , Proteínas de Ligação a Telômeros/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , DNA Helicases/genética , Reparo do DNA , Mutação , Sequências Repetitivas de Ácido Nucleico , Telomerase/genética , Telômero/química , Encurtamento do TelômeroRESUMO
The repair of DNA double-strand breaks by recombination is key to the maintenance of genome integrity in all living organisms. Recombination can however generate mutations and chromosomal rearrangements, making the regulation and the choice of specific pathways of great importance. In addition to end-joining through non-homologous recombination pathways, DNA breaks are repaired by two homology-dependent pathways that can be distinguished by their dependence or not on strand invasion catalysed by the RAD51 recombinase. Working with the plant Arabidopsis thaliana, we present here an unexpected role in recombination for the Arabidopsis RAD51 paralogues XRCC2, RAD51B and RAD51D in the RAD51-independent single-strand annealing pathway. The roles of these proteins are seen in spontaneous and in DSB-induced recombination at a tandem direct repeat recombination tester locus, both of which are unaffected by the absence of RAD51. Individual roles of these proteins are suggested by the strikingly different severities of the phenotypes of the individual mutants, with the xrcc2 mutant being the most affected, and this is confirmed by epistasis analyses using multiple knockouts. Notwithstanding their clearly established importance for RAD51-dependent homologous recombination, XRCC2, RAD51B and RAD51D thus also participate in Single-Strand Annealing recombination.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Proteínas Repressoras/genética , Arabidopsis/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Mutação , FenótipoRESUMO
Recombination establishes the chiasmata that physically link pairs of homologous chromosomes in meiosis, ensuring their balanced segregation at the first meiotic division and generating genetic variation. The visible manifestation of genetic crossing-overs, chiasmata are the result of an intricate and tightly regulated process involving induction of DNA double-strand breaks and their repair through invasion of a homologous template DNA duplex, catalysed by RAD51 and DMC1 in most eukaryotes. We describe here a RAD51-GFP fusion protein that retains the ability to assemble at DNA breaks but has lost its DNA break repair capacity. This protein fully complements the meiotic chromosomal fragmentation and sterility of Arabidopsis rad51, but not rad51 dmc1 mutants. Even though DMC1 is the only active meiotic strand transfer protein in the absence of RAD51 catalytic activity, no effect on genetic map distance was observed in complemented rad51 plants. The presence of inactive RAD51 nucleofilaments is thus able to fully support meiotic DSB repair and normal levels of crossing-over by DMC1. Our data demonstrate that RAD51 plays a supporting role for DMC1 in meiotic recombination in the flowering plant, Arabidopsis.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Meiose , Rad51 Recombinase/genética , Recombinases Rec A/genética , Recombinação Genética/genética , Arabidopsis , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
Using floral-dip, tumorigenesis and root callus transformation assays of both germline and somatic cells, we present here results implicating the four major non-homologous and homologous recombination pathways in Agrobacterium-mediated transformation of Arabidopsis thaliana. All four single mutant lines showed similar mild reductions in transformability, but knocking out three of four pathways severely compromised Agrobacterium-mediated transformation. Although integration of T-DNA into the plant genome is severely compromised in the absence of known DNA double-strand break repair pathways, it does still occur, suggesting the existence of other pathways involved in T-DNA integration. Our results highlight the functional redundancy of the four major plant recombination pathways in transformation, and provide an explanation for the lack of strong effects observed in previous studies on the roles of plant recombination functions in transformation.
Assuntos
Agrobacterium tumefaciens/genética , Arabidopsis/genética , Genoma de Planta/genética , Arabidopsis/microbiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Bacteriano/genética , DNA de Plantas/genética , Flores/genética , Flores/microbiologia , Técnicas de Inativação de Genes , Técnicas de Transferência de Genes , Genes Reporter , Vetores Genéticos , Mutação , Raízes de Plantas/genética , Raízes de Plantas/microbiologia , Tumores de Planta , Plantas Geneticamente Modificadas , Recombinação Genética , Transformação GenéticaRESUMO
During meiosis homologous chromosomes pair, recombine, and synapse, thus ensuring accurate chromosome segregation and the halving of ploidy necessary for gametogenesis. The processes permitting a chromosome to pair only with its homologue are not fully understood, but successful pairing of homologous chromosomes is tightly linked to recombination. In Arabidopsis thaliana, meiotic prophase of rad51, xrcc3, and rad51C mutants appears normal up to the zygotene/pachytene stage, after which the genome fragments, leading to sterility. To better understand the relationship between recombination and chromosome pairing, we have analysed meiotic chromosome pairing in these and in dmc1 mutant lines. Our data show a differing requirement for these proteins in pairing of centromeric regions and chromosome arms. No homologous pairing of mid-arm or distal regions was observed in rad51, xrcc3, and rad51C mutants. However, homologous centromeres do pair in these mutants and we show that this does depend upon recombination, principally on DMC1. This centromere pairing extends well beyond the heterochromatic centromere region and, surprisingly, does not require XRCC3 and RAD51C. In addition to clarifying and bringing the roles of centromeres in meiotic synapsis to the fore, this analysis thus separates the roles in meiotic synapsis of DMC1 and RAD51 and the meiotic RAD51 paralogs, XRCC3 and RAD51C, with respect to different chromosome domains.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis , Proteínas de Ciclo Celular/genética , Pareamento Cromossômico/genética , Rad51 Recombinase/genética , Recombinases Rec A/genética , Recombinação Genética , Arabidopsis/genética , Centrômero/genética , Segregação de Cromossomos , Cromossomos/genética , Fragmentação do DNA , Eucromatina/genética , Heterocromatina , Meiose/genética , Proteínas Mutantes/genética , Infertilidade das Plantas , Recombinação Genética/genéticaRESUMO
Homologous recombination is key to the maintenance of genome integrity and the creation of genetic diversity. At the mechanistic level, recombination involves the invasion of a homologous DNA template by broken DNA ends, repair of the break and exchange of genetic information between the two DNA molecules. Invasion of the template in eukaryotic cells is catalysed by the RAD51 and DMC1 recombinases, assisted by a number of accessory proteins, including the RAD51 paralogues. Eukaryotic genomes encode a variable number of RAD51 paralogues, ranging from two in yeast to five in animals and plants. The RAD51 paralogues form at least two distinct protein complexes, believed to play roles in the assembly and stabilization of the RAD51-DNA nucleofilament. Somatic recombination assays and immunocytology confirm that the three 'non-meiotic' paralogues of Arabidopsis, RAD51B, RAD51D and XRCC2, are involved in somatic homologous recombination, and that they are not required for the formation of radioinduced RAD51 foci. Given the presence of all five proteins in meiotic cells, the apparent absence of a meiotic role for RAD51B, RAD51D and XRCC2 is surprising, and perhaps simply the result of a more subtle meiotic phenotype in the mutants. Analysis of meiotic recombination confirms this, showing that the absence of XRCC2, and to a lesser extent RAD51B, but not RAD51D, increases rates of meiotic crossing over. The roles of RAD51B and XRCC2 in recombination are thus not limited to mitotic cells.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Rad51 Recombinase/genética , Animais , Arabidopsis/efeitos dos fármacos , Bleomicina/farmacologia , Núcleo Celular/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , DNA de Plantas/farmacologia , Mutação INDEL , Meiose/genética , Mitose/genética , Fenótipo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genéticaRESUMO
The ends of linear eukaryotic chromosomes are hidden in nucleoprotein structures called telomeres, and loss of the telomere structure causes inappropriate repair, leading to severe karyotypic and genomic instability. Although it has been shown that DNA damaging agents activate a DNA damage response (DDR), little is known about the signaling of dysfunctional plant telomeres. We show that absence of telomerase in Arabidopsis thaliana elicits an ATAXIA-TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR)-dependent DDR at telomeres, principally through ATM. By contrast, telomere dysfunction induces an ATR-dependent response in telomeric Conserved telomere maintenance component1 (Ctc1)-Suppressor of cdc thirteen (Stn1)-Telomeric pathways in association with Stn1 (CST)-complex mutants. These results uncover a new role for the CST complex in repressing the ATR-dependent DDR pathway in plant cells and show that plant cells use two different DNA damage surveillance pathways to signal telomere dysfunction. The absence of either ATM or ATR in ctc1 and stn1 mutants significantly enhances developmental and genome instability while reducing stem cell death. These data thus give a clear illustration of the action of ATM/ATR-dependent programmed cell death in maintaining genomic integrity through elimination of genetically unstable cells.