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

The maintenance of genome integrity over cell divisions is critical for plant development and the correct transmission of genetic information to the progeny. A key factor involved in this process is the STRUCTURAL MAINTENANCE OF CHROMOSOME5 (SMC5) and SMC6 (SMC5/6) complex, related to the cohesin and condensin complexes that control sister chromatid alignment and chromosome condensation, respectively. Here, we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the SMC5/6 complex in Arabidopsis (Arabidopsis thaliana). NSE4A is expressed in meristems and accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a mutants produce abnormal seeds, with noncellularized endosperm and embryos that maximally develop to the heart or torpedo stage. This phenotype resembles the defects in cohesin and condensin mutants and suggests a role for all three SMC complexes in differentiation during seed development. By contrast, NSE4B is expressed in only a few cell types, and loss-of-function mutants do not have any obvious abnormal phenotype. In summary, our study shows that the NSE4A subunit of the SMC5-SMC6 complex is essential for DNA damage repair in somatic tissues and plays a role in plant reproduction.

• MeSH
• Arabidopsis embryologie   genetika   imunologie   MeSH
• duplikace genu MeSH
• genom rostlinný MeSH
• oprava DNA * genetika   MeSH
• podjednotky proteinů metabolismus   MeSH
• poškození DNA * genetika   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• pyl genetika   MeSH
• regulace genové exprese u rostlin MeSH
• semena rostlinná genetika   metabolismus   MeSH
• transkriptom genetika   MeSH
• upregulace genetika   MeSH
• vajíčko rostlin genetika   MeSH
• vazba proteinů MeSH
• vývojová regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• At1g51130 protein, Arabidopsis MeSH Prohlížeč
• podjednotky proteinů MeSH
• proteiny buněčného cyklu MeSH
• proteiny huseníčku MeSH

Chromosome organization, dynamics and stability are required for successful passage through cellular generations and transmission of genetic information to offspring. The key components involved are Structural maintenance of chromosomes (SMC) complexes. Cohesin complex ensures proper chromatid alignment, condensin complex chromosome condensation and the SMC5/6 complex is specialized in the maintenance of genome stability. Here we summarize recent knowledge on the composition and molecular functions of SMC5/6 complex. SMC5/6 complex was originally identified based on the sensitivity of its mutants to genotoxic stress but there is increasing number of studies demonstrating its roles in the control of DNA replication, sister chromatid resolution and genomic location-dependent promotion or suppression of homologous recombination. Some of these functions appear to be due to a very dynamic interaction with cohesin or other repair complexes. Studies in Arabidopsis indicate that, besides its canonical function in repair of damaged DNA, the SMC5/6 complex plays important roles in regulating plant development, abiotic stress responses, suppression of autoimmune responses and sexual reproduction.

• Klíčová slova
• DNA damage repair, SMC5/6, Structural maintenance of chromosomes, chromatin, chromosomes, genome stability,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Research in algae usually focuses on the description and characterization of morpho-and phenotype as a result of adaptation to a particular habitat and its conditions. To better understand the evolution of lineages we characterized responses of filamentous streptophyte green algae of the genera Klebsormidium and Zygnema, and of a land plant-the moss Physcomitrellapatens-to genotoxic stress that might be relevant to their environment. We studied the induction and repair of DNA double strand breaks (DSBs) elicited by the radiomimetic drug bleomycin, DNA single strand breaks (SSB) as consequence of base modification by the alkylation agent methyl methanesulfonate (MMS) and of ultra violet (UV)-induced photo-dimers, because the mode of action of these three genotoxic agents is well understood. We show that the Klebsormidium and Physcomitrella are similarly sensitive to introduced DNA lesions and have similar rates of DSBs repair. In contrast, less DNA damage and higher repair rate of DSBs was detected in Zygnema, suggesting different mechanisms of maintaining genome integrity in response to genotoxic stress. Nevertheless, contrary to fewer detected lesions is Zygnema more sensitive to genotoxic treatment than Klebsormidium and Physcomitrella.

• Klíčová slova
• DNA damage and repair, Klebsormidium, Physcomitrella patens, Zygnema, bleomycin, methyl methanesulfonate, ultraviolet light,
• Publikační typ
• časopisecké články MeSH

A comparative approach in biology is needed to assess the universality of rules governing this discipline. In plant telomere research, most of the key principles were established based on studies in only single model plant, Arabidopsis thaliana. These principles include the absence of telomere shortening during plant development and the corresponding activity of telomerase in dividing (meristem) plant cells. Here we examine these principles in Physcomitrella patens as a representative of lower plants. To follow telomerase expression, we first characterize the gene coding for the telomerase reverse transcriptase subunit PpTERT in P. patens, for which only incomplete prediction has been available so far. In protonema cultures of P. patens, growing by filament apical cell division, the proportion of apical (dividing) cells was quantified and telomere length, telomerase expression and activity were determined. Our results show telomere stability and demonstrate proportionality of telomerase activity and expression with the number of apical cells. In addition, we analyze telomere maintenance in mre11, rad50, nbs1, ku70 and lig4 mutants of P. patens and compare the impact of these mutations in double-strand-break (DSB) repair pathways with earlier observations in corresponding A. thaliana mutants. Telomere phenotypes are absent and DSB repair kinetics is not affected in P. patens mutants for DSB factors involved in non-homologous end joining (NHEJ). This is compliant with the overall dominance of homologous recombination over NHEJ pathways in the moss, contrary to the inverse situation in flowering plants.

• MeSH
• Arabidopsis genetika   MeSH
• chromozomy rostlin genetika   MeSH
• DNA rostlinná genetika   MeSH
• dvouřetězcové zlomy DNA MeSH
• fenotyp MeSH
• fylogeneze MeSH
• homeostáza telomer genetika   MeSH
• homologní rekombinace MeSH
• mechy genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• oprava DNA * MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční seřazení MeSH
• telomerasa genetika   metabolismus   MeSH
• telomery genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• rostlinné proteiny MeSH
• telomerasa MeSH

The moss Physcomitrella patens is unique for the high frequency of homologous recombination, haploid state, and filamentous growth during early stages of the vegetative growth, which makes it an excellent model plant to study DNA damage responses. We used single cell gel electrophoresis (comet) assay to determine kinetics of response to Bleomycin induced DNA oxidative damage and single and double strand breaks in wild type and mutant lig4 Physcomitrella lines. Moreover, APT gene when inactivated by induced mutations was used as selectable marker to ascertain mutational background at nucleotide level by sequencing of the APT locus. We show that extensive repair of DSBs occurs also in the absence of the functional LIG4, whereas repair of SSBs is seriously compromised. From analysis of induced mutations we conclude that their accumulation rather than remaining lesions in DNA and blocking progression through cell cycle is incompatible with normal plant growth and development and leads to sensitive phenotype.

• MeSH
• analýza jednotlivých buněk MeSH
• bleomycin farmakologie   MeSH
• buněčný cyklus genetika   MeSH
• haploidie * MeSH
• homologní rekombinace genetika   MeSH
• mechy genetika   růst a vývoj   MeSH
• mutace MeSH
• mutageneze genetika   MeSH
• mutageny farmakologie   MeSH
• oprava DNA genetika   MeSH
• oxidační stres účinky léků   MeSH
• poškození DNA účinky léků   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny biosyntéza   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bleomycin MeSH
• mutageny MeSH
• rostlinné proteiny MeSH