The Phenotype of Physcomitrium patens SMC6 Mutant with Interrupted Hinge Interactions

<b>Background/Objectives</b>: The Structural Maintenance of Chromosomes (SMC) proteins form essential heterocomplexes for the preservation of DNA structure and its functions, and hence cell viability. The SMC5/6 dimer is assembled by direct interactions of ATP heads via the kleisin NSE4 bridge and by SMC hinges. The structure might be interrupted by a single point mutation within a conserved motif of the SMC6-hinge. We describe the phenomena associated with the impairment of the SMC5/6 complex with morphology, repair of DNA double strand breaks (DSB), mutagenesis, recombination and gene targeting (GT) in the moss <i>Physcomitrium patens</i> (<i>P. patens</i>). <b>Methods</b>: Using CRISPR/Cas9-directed oligonucleotide replacement, we have introduced two close G to R point mutations in the hinge domain of SMC6 of <i>P. patens</i> and show that both mutations are not toxic and allow viability of mutant lines. <b>Results</b>: The <i>G514R</i> mutation fully prevents the interaction of SMC6 not only with SMC5, but also with NSE5 and NSE6, while the mutation at <i>G517R</i> has no effect. The <i>Ppsmc6_G514R</i> line has aberrant morphology, spontaneous and bleomycin-induced mutagenesis, and maintenance of the number of rDNA copies. The most unique feature is the interference with gene targeting (GT), which is completely abolished. In contrast, the <i>Ppsmc6_G517R</i> line is close to WT in many aspects. Surprisingly, both mutations have no direct effect on the rate of DSB repair in dividing and differentiated cells. <b>Conclusions:</b> Abolished interactions of SMC6 with SMC5 and NSE5,6 partners, which allow DSB repair, but impair other repair and recombination functions, suggests also regulatory role for SMC6.