Biology subjects

Palecek, J. J.

Publications and source records attributed to Palecek, J. J..

8 recordsLinked to original sources

bioRxiv2026

La protein binding to telomerase RNA supports an evolutionary relationship between plant and ciliate telomerase pathways

The Arabidopsis thaliana La1 (AtLa1) protein is a member of the genuine La family of RNA biogenesis proteins, which are structurally similar to the La-resembling protein 7 (LARP7) family. LARP7 proteins participate in the biogenesis of the telomerase ribonucleoprotein complex in model systems, but are absent in plants. We show that AtLa1 binds to telomerase RNA in a manner reminiscent of the Tetrahymena LARP7 protein p65. Classical in vitro methods and microscale thermophoresis (MST) were used to specify the molecular structures involved in this multi-surface interaction. AtLa1 also enhances the binding of TR to the telomerase reverse transcriptase RNA binding domain. We therefore propose that biogenesis of telomerase RNA in plants and ciliates is achieved by a similar pathway, differing in the employment of genuine La or LARP7-like proteins, respectively. We also report that the domain of unknown function (DUF3223, DeCL) found in the AtLa1 protein binding partner, Domino, is an RNA binding domain with modest TR-binding capacity. This domain is also found in plant and ciliate proteins, including plant polymerases IV/V and the Tetrahymena La protein Mlp1. Together, these suggest that RNA biogenesis pathways in plants and ciliates have a conserved evolutionary relationship, with parallels between their La proteins.

biophysics↗

bioRxiv2025

Homologous recombination mutant lethality differs between h- and h+ Schizosaccharomyces pombe strains due to mat1 heterochromatin

Homologous recombination (HR) is generally considered dispensable in yeast and vertebrates, yet mounting evidence indicates that its essentiality depends on cellular context. Here, we dissect the basis of this context dependency in Schizosaccharomyces pombe. In the homothallic h90 strain, regarded as wild type, mating-type switching (MTS) occurs every other cell division and requires HR to repair programmed double-strand breaks (DSBs) at the mat1 locus. We show that the widely used heterothallic h-S strain is likewise dependent on HR for viability. HR-deficient h-S mutants (rad51{Delta}, rad52{Delta}, or rad54{Delta}), still frequently employed in the literature, survive only when carrying secondary suppressor mutations that abolish mat1 DSB formation, such as smt-0, swi1{Delta}, or fml1{Delta}. In contrast, HR is dispensable in the h+N strain, where duplication of the mat2/3 region into mat1 introduces the cenH and REIII elements. These elements nucleate H3K9 methylation and heterochromatin spreading across the imprint site, blocking imprintosome recruitment and thereby preventing both imprinting and DSB formation. Disruption of this heterochromatin, via deletion of cenH or key chromatin modifiers, restores DSB formation in h+N cells and reinstates HR essentiality in the absence of the Clr4 methyltransferase. Collectively, our findings demonstrate that HR is indispensable for S. pombe survival due to its critical role in repairing mat1 DSBs, except under genetic or epigenetic conditions that suppress their formation.

molecular biology↗

bioRxiv2025

The C-terminal SUMOylation-dependent regulation of αKNL2 governs its centromere targeting and interaction with CENH3

The centromere is a specialized domain that facilitates chromosome segregation during mitosis and serves as the site for kinetochore formation. KINETOCHORE NULL2 (KNL2) is essential for the recognition and loading of the centromeric histone H3 variant, CENH3, to centromeres. A yeast two-hybrid screen for KNL2 interactors identified components of the SUMOylation pathway. However, the role of KNL2 SUMOylation in Arabidopsis has not yet been determined. In this study, we demonstrated that the C-terminal part of KNL2 interacts with SUMO3 and ULP1d, as shown by BiFC and co-immunoprecipitation assays. Bioinformatic and functional analysis identified three SUMOylation and two SUMO-interacting motif (SIM) sites in the C-terminal region of KNL2, which are critical for growth, fertility, and chromosome alignment. Of the three SUMOylation sites, Lys474 and Lys511 were the most critical for the centromeric localization of KNL2, underscoring the importance of KNL2 SUMOylation for its function. Additionally, both in vitro and in vivo assays showed that KNL2-C undergoes SUMOylation by SUMO1 or SUMO3. The SUMO protease mutant, ulp1d-2 led to the slight accumulation of SUMOylated KNL2 in Arabidopsis. We further showed that SUMOylation of KNL2 promotes its binding to CENH3 and controls protein stability. Our findings show that C-terminal SUMOylation of KNL2 is crucial for its centromeric localization, interaction with CENH3, and kinetochore assembly, emphasizing the significance of post-translational modifications in chromosome segregation and cell division in plants.

molecular biology↗

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

SAGA histone acetyltransferase module facilitates chromatin accessibility to SMC5/6

Structural Maintenance of Chromosomes (SMC) complexes are molecular machines driving chromatin organization at higher levels. In eukaryotes, three SMC complexes (cohesin, condensin, and SMC5/6) play key roles in cohesion, condensation, replication, transcription and DNA repair. Here, we performed a genetic screen in fission yeast to identify novel factors required for SMC5/6 binding to DNA. We identified 79 genes of which histone acetyltransferases (HATs) were the most represented. Genetic and phenotypic analyses suggested a particularly strong functional relationship between the SMC5/6 and SAGA complexes. Furthermore, several SMC5/6 subunits physically interacted with SAGA HAT module components Gcn5 and Ada2. As Gcn5-dependent acetylation facilitates the accessibility of chromatin to DNA repair proteins, we first analysed the formation of DNA damage-induced SMC5/6 foci in the {Delta}gcn5 mutant. The SMC5/6 foci formed normally in {Delta}gcn5, suggesting SAGA-independent SMC5/6 localization to DNA-damaged sites. Next, we used Nse4-FLAG chromatin-immunoprecipitation (ChIP-seq) analysis in unchallenged cells to assess SMC5/6 distribution. A significant portion of SMC5/6 accumulated within gene regions in wild-type cells, which was reduced in {Delta}gcn5 and {Delta}ada2 mutants. The drop in SMC5/6 levels was also observed in gcn5-E191Q acetyltransferase-dead mutant. Altogether, our data suggest that the SAGA HAT module may facilitate chromatin accessibility to SMC5/6 at gene regions. Author SummaryGenomes of all eukaryotes must be folded and packed into their relatively small nuclear spaces. Histones first pack free genomic DNA into nucleosomes and their arrays. Other complexes like histone modifiers and remodelers can regulate nucleosome positions and their packing within chromatin fibres. They assist in the relative opening or condensation of chromatin fibres and facilitate their accessibility to DNA-binding proteins. The highly conserved Structural Maintenance of Chromosomes (SMC) complexes (cohesin, condensin, and SMC5/6) compact further chromatin fibres at higher levels. These molecular machines can loop chromatin fibres, which need access to segments of free DNA for their physical binding to DNA. Here, we studied genetic and physical interactions between histone-modifying SAGA complex and SMC5/6. We show that the SAGA histone acetyltransferase module may facilitate chromatin access to SMC5/6.

genetics↗

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↗

bioRxiv2019

A role of the Nse4 kleisin and Nse1/Nse3 KITE subunits in the ATPase cycle of SMC5/6

The SMC (Structural Maintenance of Chromosomes) complexes are composed of SMC dimers, kleisin and kleisin-interacting subunits. Mutual interactions of these subunits constitute the basal architecture of the SMC complexes. Particularly, terminal domains of the kleisin subunit bridge the SMC head domains of the SMC molecules. Binding of ATP molecules to the heads and their hydrolysis alter the shape of long SMC molecules (from rod-like to open-ring shapes) and power them with motor activity. The kleisin-interacting (HAWK or KITE) subunits bind kleisin linker regions and regulate such dynamic activities.\n\nWe developed new systems to follow the interactions between SMC5/6 subunits and stability of the SMC5/6 complexes. 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 part increases stability of the ATP-free SMC5/6 complex. In contrast, binding of ATP to the SMC5/6 complex containing KITE subunits significantly decreases its stability. Elongation of the Nse4 linker partially suppresses 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.\n\nAUTHOR SUMMARYThe SMC5/6 complex is member of the Structural maintenance of chromosomes (SMC) family, key organizers of both prokaryotic and eukaryotic genomes. Their architecture and dynamics (driven by ATP binding and hydrolysis) are essential for cellular processes, like chromatin segregation, condensation, replication and repair. In this paper, we described conserved mode of the Nse4 kleisin subunit binding to the SMC6 (similar to cohesin and condensin) and its bridging role. Furthermore, we showed different impact of the binding of the Nse1-Nse3 KITE subunits to the Nse4 kleisin bridge. Our study suggested that the KITE proteins modulate the stability of the SMC5/6 complex, depending on its binding and hydrolysis of ATP. Our findings uncover molecular mechanisms underlying dynamics of the SMC5/6 complexes.

genetics↗