SMC5/6 is a highly conserved protein complex related to cohesin and condensin, which are the key components of higher-order chromatin structures. The SMC5/6 complex is essential for proliferation in yeast and is involved in replication fork stability and processing. However, the precise mechanism of action of SMC5/6 is not known. Here we present evidence that the NSE1/NSE3/NSE4 sub-complex of SMC5/6 binds to double-stranded DNA without any preference for DNA-replication/recombination intermediates. Mutations of key basic residues within the NSE1/NSE3/NSE4 DNA-binding surface reduce binding to DNA in vitro. Their introduction into the Schizosaccharomyces pombe genome results in cell death or hypersensitivity to DNA damaging agents. Chromatin immunoprecipitation analysis of the hypomorphic nse3 DNA-binding mutant shows a reduced association of fission yeast SMC5/6 with chromatin. Based on our results, we propose a model for loading of the SMC5/6 complex onto the chromatin.
The SMC (Structural Maintenance of Chromosomes) complexes are composed of SMC dimers, kleisin and kleisin-interacting (HAWK or KITE) subunits. Mutual interactions of these subunits constitute the basal architecture of the SMC complexes. In addition, binding of ATP molecules to the SMC subunits and their hydrolysis drive dynamics of these complexes. Here, we developed new systems to follow the interactions between SMC5/6 subunits and the relative stability of the complex. 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 increased stability of the ATP-free SMC5/6 complex. In contrast, binding of ATP to SMC5/6 containing KITE subunits significantly decreased its stability. Elongation of the Nse4 linker partially suppressed 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.
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.
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