Nejvíce citovaný článek - PubMed ID 31036599
The SMC5/6 Complex Subunit NSE4A Is Involved in DNA Damage Repair and Seed Development
DNA-protein cross-links (DPCs) are highly toxic DNA lesions consisting of proteins covalently attached to chromosomal DNA. Unrepaired DPCs physically block DNA replication and transcription. Three DPC repair pathways have been identified in Arabidopsis (Arabidopsis thaliana) to date: the endonucleolytic cleavage of DNA by the structure-specific endonuclease MUS81; proteolytic degradation of the crosslinked protein by the metalloprotease WSS1A; and cleavage of the cross-link phosphodiester bonds by the tyrosyl phosphodiesterases TDP1 and TDP2. Here we describe the evolutionary conserved STRUCTURAL MAINTENANCE OF CHROMOSOMEs SMC5/6 complex as a crucial component involved in DPC repair. We identified multiple alleles of the SMC5/6 complex core subunit gene SMC6B via a forward-directed genetic screen designed to identify the factors involved in the repair of DPCs induced by the cytidine analog zebularine. We monitored plant growth and cell death in response to DPC-inducing chemicals, which revealed that the SMC5/6 complex is essential for the repair of several types of DPCs. Genetic interaction and sensitivity assays showed that the SMC5/6 complex works in parallel to the endonucleolytic and proteolytic pathways. The repair of zebularine-induced DPCs was associated with SMC5/6-dependent SUMOylation of the damage sites. Thus, we present the SMC5/6 complex as an important factor in plant DPC repair.
- MeSH
- Arabidopsis * genetika metabolismus MeSH
- DNA metabolismus MeSH
- oprava DNA genetika MeSH
- poškození DNA MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- proteiny metabolismus MeSH
- sumoylace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
- proteiny buněčného cyklu MeSH
- proteiny MeSH
Chromatin-based processes are essential for cellular functions. Structural maintenance of chromosomes (SMCs) are evolutionarily conserved molecular machines that organize chromosomes throughout the cell cycle, mediate chromosome compaction, promote DNA repair, or control sister chromatid attachment. The SMC5/6 complex is known for its pivotal role during the maintenance of genome stability. However, a dozen recent plant studies expanded the repertoire of SMC5/6 complex functions to the entire plant sexual reproductive phase. The SMC5/6 complex is essential in meiosis, where its activity must be precisely regulated to allow for normal meiocyte development. Initially, it is attenuated by the recombinase RAD51 to allow for efficient strand invasion by the meiosis-specific recombinase DMC1. At later stages, it is essential for the normal ratio of interfering and non-interfering crossovers, detoxifying aberrant joint molecules, preventing chromosome fragmentation, and ensuring normal chromosome/sister chromatid segregation. The latter meiotic defects lead to the production of diploid male gametes in Arabidopsis SMC5/6 complex mutants, increased seed abortion, and production of triploid offspring. The SMC5/6 complex is directly involved in controlling normal embryo and endosperm cell divisions, and pioneer studies show that the SMC5/6 complex is also important for seed development and normal plant growth in cereals.
- Klíčová slova
- SMC5/6 complex, fertility, genome stability, meiosis, polyploidy, reproductive development, seed,
- MeSH
- Arabidopsis * genetika metabolismus MeSH
- chromatidy metabolismus MeSH
- meióza MeSH
- oprava DNA MeSH
- proteiny buněčného cyklu * genetika metabolismus MeSH
- rekombinasy genetika MeSH
- rozmnožování genetika MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- proteiny buněčného cyklu * MeSH
- rekombinasy MeSH
Loss of genome stability leads to reduced fitness, fertility and a high mutation rate. Therefore, the genome is guarded by the pathways monitoring its integrity and neutralizing DNA lesions. To analyze the mechanism of DNA damage induction by cytidine analog zebularine, we performed a forward-directed suppressor genetic screen in the background of Arabidopsis thaliana zebularine-hypersensitive structural maintenance of chromosomes 6b (smc6b) mutant. We show that smc6b hypersensitivity was suppressed by the mutations in EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3 (ENT3), DNA METHYLTRANSFERASE 1 (MET1) and DECREASE IN DNA METHYLATION 1 (DDM1). Superior resistance of ent3 plants to zebularine indicated that ENT3 is likely necessary for the import of the drug to the cells. Identification of MET1 and DDM1 suggested that zebularine induces DNA damage by interference with the maintenance of CG DNA methylation. The same holds for structurally similar compounds 5-azacytidine and 2-deoxy-5-azacytidine. Based on our genetic and biochemical data, we propose that zebularine induces enzymatic DNA-protein crosslinks (DPCs) of MET1 and zebularine-containing DNA in Arabidopsis, which was confirmed by native chromatin immunoprecipitation experiments. Moreover, zebularine-induced DPCs accumulate preferentially in 45S rDNA chromocenters in a DDM1-dependent manner. These findings open a new avenue for studying genome stability and DPC repair in plants.
- MeSH
- Arabidopsis MeSH
- cytidin analogy a deriváty toxicita MeSH
- DNA vazebné proteiny genetika MeSH
- DNA-(cytosin-5-)methyltransferasa genetika MeSH
- heterochromatin účinky léků metabolismus MeSH
- léková rezistence MeSH
- membránové transportní proteiny genetika MeSH
- mutace MeSH
- mutageny toxicita MeSH
- proteiny buněčného cyklu genetika MeSH
- proteiny huseníčku genetika MeSH
- RNA ribozomální účinky léků genetika MeSH
- transkripční faktory genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- AT4G05120 protein, Arabidopsis MeSH Prohlížeč
- cytidin MeSH
- DDM1 protein, Arabidopsis MeSH Prohlížeč
- DNA vazebné proteiny MeSH
- DNA-(cytosin-5-)methyltransferasa MeSH
- heterochromatin MeSH
- membránové transportní proteiny MeSH
- MET1 protein, Arabidopsis MeSH Prohlížeč
- mutageny MeSH
- proteiny buněčného cyklu MeSH
- proteiny huseníčku MeSH
- pyrimidin-2-one beta-ribofuranoside MeSH Prohlížeč
- RNA ribozomální MeSH
- RNA, ribosomal, 45S MeSH Prohlížeč
- SMC6B protein, Arabidopsis MeSH Prohlížeč
- transkripční faktory MeSH
Kleisin NSE4 and circular form of SMC5/6 is indispensable for DSB repair and necessary for gene targeting but is not enough for recovery of cells from DNA damage in Physcomitrella. Structural maintenance of chromosomes (SMC) complexes are involved in cohesion, condensation and maintenance of genome stability. Based on the sensitivity of mutants to genotoxic stress the SMC5/6 complex is thought to play a prominent role in DNA stabilization during repair by tethering DNA at the site of lesion by a heteroduplex of SMC5 and SMC6 encircled with non-SMC components NSE1, NSE3 and kleisin NSE4. In this study, we tested how formation of the SMC5/6 circular structure affects mutant sensitivity to DNA damage, kinetics of DSB repair and gene targeting. In the moss Physcomitrella (Physcomitrium patens), SMC6 and NSE4 are essential single copy genes and this is why we used blocking of transcription to reveal their mutated phenotype. Even slight reduction of transcript levels by dCas9 binding was enough to obtain stable lines with severe DSB repair defects and specific bleomycin sensitivity. We show that survival after bleomycin or MMS treatment fully depends on active SMC6, whereas attenuation of NSE4 has little or negligible effect. We conclude that circularization of SMC5/6 provided by the kleisin NSE4 is indispensable for the DSB repair, nevertheless there are other functions associated with the SMC5/6 complex, which are critical to survive DNA damage.
- Klíčová slova
- Comet assay, DNA repair, Gene targeting, NSE4 kleisin, Physcomitrella, Physcomitrium patens, SMC5/6 complex, dCas9,
- MeSH
- DNA rostlinná genetika metabolismus MeSH
- dvouřetězcové zlomy DNA * MeSH
- fylogeneze MeSH
- genotyp MeSH
- mechy genetika metabolismus MeSH
- multiproteinové komplexy genetika metabolismus MeSH
- mutace MeSH
- oprava DNA * MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- poškození DNA * MeSH
- proteiny buněčného cyklu klasifikace genetika metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny klasifikace genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA rostlinná MeSH
- multiproteinové komplexy MeSH
- proteiny buněčného cyklu MeSH
- rostlinné proteiny MeSH
Polyploidization is a common phenomenon in the evolution of flowering plants. However, only a few genes controlling polyploid genome stability, fitness, and reproductive success are known. Here, we studied the effects of loss-of-function mutations in NSE2 and NSE4A subunits of the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex in autotetraploid Arabidopsis thaliana plants. The diploid nse2 and nse4a plants show partially reduced fertility and produce about 10% triploid offspring with two paternal and one maternal genome copies. In contrast, the autotetraploid nse2 and nse4a plants were almost sterile and produced hexaploid and aneuploid progeny with the extra genome copies or chromosomes coming from both parents. In addition, tetraploid mutants had more severe meiotic defects, possibly due to the presence of four homologous chromosomes instead of two. Overall, our study suggests that the SMC5/6 complex is an important player in the maintenance of tetraploid genome stability and that autotetraploid Arabidopsis plants have a generally higher frequency of but also higher tolerance for aneuploidy compared to diploids.
- Klíčová slova
- NSE2, SMC5/6 complex, genome stability, meiosis, polyploidy, seed development,
- Publikační typ
- časopisecké články MeSH
Structural maintenance of chromosome 5/6 (SMC5/6) complex is a crucial factor for preserving genome stability. Here, we show that mutants for several Arabidopsis (Arabidopsis thaliana) SMC5/6 complex subunits produce triploid offspring. This phenotype is caused by a meiotic defect leading to the production of unreduced male gametes. The SMC5/6 complex mutants show an absence of chromosome segregation during the first and/or the second meiotic division, as well as a partially disorganized microtubule network. Importantly, although the SMC5/6 complex is partly required for the repair of SPO11-induced DNA double-strand breaks, the nonreduction described here is SPO11-independent. The measured high rate of ovule abortion suggests that, if produced, such defects are maternally lethal. Upon fertilization with an unreduced pollen, the unbalanced maternal and paternal genome dosage in the endosperm most likely causes seed abortion observed in several SMC5/6 complex mutants. In conclusion, we describe the function of the SMC5/6 complex in the maintenance of gametophytic ploidy in Arabidopsis.
The SMC (Structural Maintenance of Chromosomes) complexes are composed of SMC dimers, kleisin and kleisin-interacting (HAWK or KITE) subunits. Mutual interactions of these subunits constitute the basal architecture of the SMC complexes. In addition, binding of ATP molecules to the SMC subunits and their hydrolysis drive dynamics of these complexes. Here, we developed new systems to follow the interactions between SMC5/6 subunits and the relative stability of the complex. First, we show that the N-terminal domain of the Nse4 kleisin molecule binds to the SMC6 neck and bridges it to the SMC5 head. Second, binding of the Nse1 and Nse3 KITE proteins to the Nse4 linker increased stability of the ATP-free SMC5/6 complex. In contrast, binding of ATP to SMC5/6 containing KITE subunits significantly decreased its stability. Elongation of the Nse4 linker partially suppressed instability of the ATP-bound complex, suggesting that the binding of the KITE proteins to the Nse4 linker constrains its limited size. Our data suggest that the KITE proteins may shape the Nse4 linker to fit the ATP-free complex optimally and to facilitate opening of the complex upon ATP binding. This mechanism suggests an important role of the KITE subunits in the dynamics of the SMC5/6 complexes.
- MeSH
- adenosintrifosfatasy metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- makromolekulární látky metabolismus MeSH
- mutageneze cílená MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- Schizosaccharomyces pombe - proteiny genetika metabolismus MeSH
- Schizosaccharomyces genetika metabolismus MeSH
- sekvenční seřazení MeSH
- techniky dvojhybridového systému MeSH
- transportní proteiny genetika metabolismus MeSH
- vazba proteinů genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adenosintrifosfatasy MeSH
- jaderné proteiny MeSH
- makromolekulární látky MeSH
- Nse1 protein, S pombe MeSH Prohlížeč
- Nse3 protein, S pombe MeSH Prohlížeč
- Nse4 protein, S pombe MeSH Prohlížeč
- proteiny buněčného cyklu MeSH
- Schizosaccharomyces pombe - proteiny MeSH
- Smc5 protein, S pombe MeSH Prohlížeč
- smc6 protein, S pombe MeSH Prohlížeč
- transportní proteiny MeSH
The SMC 5/6 complex together with cohesin and condensin is a member of the structural maintenance of chromosome (SMC) protein family. In non-plant organisms SMC5/6 is engaged in DNA repair, meiotic synapsis, genome organization and stability. In plants, the function of SMC5/6 is still enigmatic. Therefore, we analyzed the crucial δ-kleisin component NSE4 of the SMC5/6 complex in the model plant Arabidopsis thaliana. Two functional conserved Nse4 paralogs (Nse4A and Nse4B) are present in A. thaliana, which may have evolved via gene subfunctionalization. Due to its high expression level, Nse4A seems to be the more essential gene, whereas Nse4B appears to be involved mainly in seed development. The morphological characterization of A. thaliana T-DNA mutants suggests that the NSE4 proteins are essential for plant growth and fertility. Detailed investigations in wild-type and the mutants based on live cell imaging of transgenic GFP lines, fluorescence in situ hybridization (FISH), immunolabeling and super-resolution microscopy suggest that NSE4A acts in several processes during plant development, such as mitosis, meiosis and chromatin organization of differentiated nuclei, and that NSE4A operates in a cell cycle-dependent manner. Differential response of NSE4A and NSE4B mutants after induced DNA double strand breaks (DSBs) suggests their involvement in DNA repair processes.
- Klíčová slova
- Arabidopsis thaliana, NSE4 δ-kleisin, SMC5/6 complex, meiosis, mitosis, nucleus, phylogeny, super-resolution microscopy,
- Publikační typ
- časopisecké články MeSH
