Adamus, M. | bioRxiv Science

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↗