The phenotype of SMC6_G514R hinge mutant of Physcomitrium patents

Abstract

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.