Characterization of the conserved features of the NSE6 subunit of the Physcomitrium patens SMC5/6 complex

Abstract

SUMMARYStructural maintenance of chromosomes (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 subunits. Additional factors, like NSE6 within SMC5/6, bind to SMC core complexes and regulate their activities. In the human HsNSE6/SLF2, we recently identified a new CANIN domain. Here we tracked down its sequence homology to lower plants, selected the bryophyte Physcomitrium patens, and analyzed PpNSE6 protein–protein interactions to explore its conservation in detail. We identified a previously unrecognized core sequence motif conserved from yeasts to humans within the NSE6 CANIN domain. This motif mediates the interaction between NSE6 and its NSE5 partner in yeasts and plants. In addition, the CANIN domain and its preceding PpNSE6 sequences bind both PpSMC5 and PpSMC6 arms. Interestingly, we mapped the PpNSE6‐binding site at the PpSMC5 arm right next to the PpNSE2‐binding surface. The position of NSE6 at SMC arms suggests its role in the regulation of SMC5/6 dynamics. Consistent with the regulatory role of NSE6 subunits, Ppnse6 mutant lines were viable and sensitive to the DNA‐damaging drug bleomycin and lost a large portion of rDNA copies. These moss mutants also exhibited reduced growth and developmental aberrations. Altogether, our data showed the conserved function of the NSE6 subunit and architecture of the SMC5/6 complex across species.

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

“…To verify our Y2H results, we expressed the PpNSE5(aa190–526) and PpNSE5(aa245–526) fragments in vitro and used them in a pull‐down assay (Figure 1d; Lelkes et al., 2023). The Stag‐PpNSE6(aa1–370) specifically precipitated PpNSE5(aa190–526), while not precipitating PpNSE5(aa245–526) (Figure 1d, lanes 3 and 9), thus confirming the Y2H results.…”

“…Recently, we characterized the moss PpNSE6 subunit of SMC5/6 and its binding to the PpNSE5 partner (Lelkes et al., 2023). Here, we complemented this analysis with a study of the binding properties of PpNSE5.…”

“…Yeast NSE5 subunits bind directly to SMC arms (Li et al., 2023; Palecek et al., 2006), while there is no evidence for the direct binding of human HsNSE5 orthologs (HsNSE5A/SLF1 or HsNSE5B/SIMC1) to SMC subunits (Adamus et al., 2020; Oravcova et al., 2022). Therefore, we tested the above Y2H constructs (Figure 1a) and other PpNSE5 fragments (Figure 2) against PpSMC5 and PpSMC6 constructs (Lelkes et al., 2023). Surprisingly, only Gal4AD‐PpNSE5(aa1–230) bound PpSMC5 and PpSMC6 (Figure 2a).…”

“…To analyze PpNSE5–PpSMC6 interaction further, we prepared different arm fragments of PpSMC6 truncated from the head‐ or hinge‐proximal end (Figure 3a; Lelkes et al., 2023). The long headless fragments Gal4BD‐PpSMC6(aa226–955) and (aa255–923) bound both Gal4AD‐PpNSE5(aa1–230) and Gal4AD‐PpNSE6(aa1–370) (Figure 3b, lanes 1 and 2).…”

Shaping up genomes: Prokaryotic roots and eukaryotic diversification of SMC complexes

“…Additionally, we selected proteins regulating these complexes, provided that they are specific (i.e., no general cell cycle regulators like PP2A, CDK1, and Aurora kinases). The selected proteins should have been characterized as a complex component in at least one organism, predominantly H. sapiens , S. cerevisiae and S. pombe 22,46 , and also T. thermophila 10,16,47,48 , P. patens 49,50 , A. thaliana 28,51 and C. merolae 18 .…”