Biology subjects

Vagnerova, R.

Publications and source records attributed to Vagnerova, R..

4 recordsLinked to original sources

bioRxiv2024

The phenotype of SMC6_G514R hinge mutant of Physcomitrium patents

The Structural Maintenance of Chromosomes (SMC) complexes play roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of SMC proteins with a unique structure consisting of an ATPase head, long arm, and hinge. The direct interaction of hinges leads to the formation of SMC heterodimer. A critical SMC6 mutation G551R interrupting the interaction of SMC5 and SMC6 hinges have been previously identified in Schizosaccharomyces pombe within a conserved motif. Using CRISPR/Cas9 directed oligonucleotide replacement, we have introduced this G to R point mutation in SMC6 of Physcomitrium patens (P. patens) at position 514 and also at position 517 of the same hinge domain. It turned out that both mutations are not toxic and do not affect the viability of established Ppsmc6_G514R and Ppsmc6_G517R lines. Since P. patens mutants with entire or partial deletion of the SMC6 gene are not viable, we compare hinge mutants with previously established mutant line with attenuated transcription by targeted binding of deactivated Cas9 nuclease (Ppsmc6_dCas). We show that mutation of G to R at position 514 fully prevents the interaction of SMC6 not only with SMC5, but also NSE5 and NSE6. Surprisingly, mutation of close residue 517 has no effect at all. The Ppsmc6_G514R line has aberrant morphology quite similar to Ppsmc6_dCas, though the absence of protonemata branching and formation of gametophores is incomplete. On the contrary, the Ppsmc6_G517R line is morphologically more or less similar to WT. Spontaneous and bleomycin-induced mutagenesis and maintenance of the number of rDNA copies in the Ppsmc6_G514R line is also similar to Ppsmc6_dCas, while Ppsmc6_G517R more or less mimics WT. The sensitivity of the Ppsmc6_G514R line to bleomycin is not as severe as that of Ppsmc6_dCas, and surprisingly, the Ppsmc6_G517R line is even less sensitive to bleomycin than WT. Moreover, both hinge mutations have no direct effect on the rate of DSB repair in dividing and differentiated cells. The most unique feature of the hinge mutants is interference with gene targeting (GT). Whilst GT efficiency of Ppsmc6_G517R and Ppsmc6_dCas when compared to WT is only slightly or moderately reduced, it is completely abolished in Ppsmc6_G514R. Based on these results, we conclude that sufficient amounts of SMC6 and its interactions are necessary for normal moss development and genome stability, such as DNA repair and rDNA maintenance. The reduced levels of SMC6 subunit, and therefore low levels of complete SMC5/6 complex, are insufficient for acute DSB repair, however, the acute DSB repair is not affected by impaired SMC6 interactions in Ppsmc6_G514R. In contrast, SMC6 inability to interact with SMC5 and other partners like NSE5 and NSE6 results in abolished GT, while low levels of SMC6 have only mild effect. These data underline importance of different aspects of SMC5/6, such as its levels or interactions.

molecular biology↗

bioRxiv2024

NSE5 subunit interacts with distant regions of the SMC arms in the Physcomitrium patens SMC5/6 complex

Structural Maintenance of Chromosome (SMC) complexes play roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of SMC proteins with a unique structure consisting of an ATPase head, long arm, and hinge. SMC complexes form long rod-like structures, which can change to ring-like and elbow-bent conformations upon binding ATP, DNA and other regulatory factors. These SMC dynamic conformational changes are involved in their loading, translocation, and DNA loop extrusion. Here, we examined the binding and role of the PpNSE5 regulatory factor of Physcomitrium patens PpSMC5/6 complex. We found that the PpNSE5 C-terminal half (aa230-505) is required for binding to its PpNSE6 partner, while the N-terminal half (aa1-230) binds PpSMC subunits. Specifically, the first 71 amino acids of PpNSE5 were required for binding to PpSMC6. Interestingly, the PpNSE5 binding required the PpSMC6 head-proximal joint region and PpSMC5 hinge-proximal arm, suggesting a long distance between binding sites on PpSMC5 and PpSMC6 arms. Given the long distance between these PpSMC sites and the size of PpNSE5, we hypothesize that PpNSE5 either links two antiparallel SMC5/6 complexes or binds one SMC5/6 in elbow-bent conformation. In addition, we generated the P. patens mutant lines (Ppnse5KO1 and Ppnse5KO2) with CRISPR/Cas9-integrated stop codons in PpNSE5. The Ppnse5KO1 mutant line with an N-terminally truncated version of PpNSE5 (starting from an alternative aaMet72) exhibited DNA repair defects while keeping a normal number of rDNA repeats. As the first 71 amino acids of PpNSE5 are required for PpSMC6 binding, our results suggest the specific role of PpNSE5-PpSMC6 interaction in DNA repair. Altogether, our study suggests that PpNSE5 binding to distant regions of the PpSMC5 and PpSMC6 arms serves a specific role in loading at DNA lesions.

molecular biology↗

bioRxiv2022

Characterization of the NSE6 subunit of the Physcomitrium patens PpSMC5/6 complex

Structural Maintenance of Chromosome (SMC) complexes are molecular machines ensuring chromatin organization at higher levels. They play direct roles in cohesion, condensation, replication, transcription and DNA repair. Their cores are composed of long-armed SMC, kleisin, and kleisin-associated KITE or HAWK subunits. Additional factors, like NSE6 within SMC5/6, bind to SMC core complexes and regulate their activities. To characterize the NSE6 subunit of moss Physcomitrium patens, we analyzed its protein-protein interactions and Ppnse6 mutant phenotypes. We identified a previously unrecognized sequence motif conserved from yeast to humans within the NSE6 CANIN domain that is required for interaction with its NSE5 partner. In addition, the CANIN domain and its preceding sequences bind and link SMC5 and SMC6 arms, suggesting its role in SMC5/6 dynamics. Both Ppnse6dCas9_3 and Ppnse6KO1_47 mutant lines exhibited reduced growth and developmental aberrations. These mutants were also sensitive to DNA-damaging drug bleomycin and lost a significant portion of rDNA copies, suggesting conserved architecture and functions of SMC5/6 complexes across species.

molecular biology↗

bioRxiv2020

The NHEJ Repair of DNA Double Strand Breaks in Physcomitrella patens Depends on the Kleisin NSE4 of the SMC5/6 Complex.

Structural maintenance of chromosomes (SMC) complexes are involve 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 imminent role in DNA stabilization during repair by encircling DNA at the site of lesion by bridging the heteroduplex of SMC5 and SMC6 by non SMC kleisin components NSE1, 3 and 4. In this study, we tested how formation of the SMC5/6 circular structure affects mutant sensitivity to genotoxic stress, kinetics of DSB repair and insertion mutagenesis. In the moss Physcomitrella 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 attenuation of transcription by dCas9 binding was enough to obtain stable lines with DSB repair defect and specific bleomycin sensitivity. Whereas survival after bleomycin or MMS treatment fully depends on active SMC6, NSE4 has little or negligible effect. We conclude that whereas circularization of SMC5/6 provided by the kleisin NSE4 is indispensable for the immediate NHEJ DSB repair response, other functions associated with SMC5/6 complex are critical to survive DNA damage.

molecular biology↗