Kralova, B. | Search bioRxiv

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

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↗