Kleisin NSE4 of the SMC5/6 complex is necessary for DNA double strand break repair, but not for recovery from DNA damage in Physcomitrella (Physcomitrium patens)

Abstract

Structural maintenance of chromosomes (SMC) complexes are involve in cohesion, condensation and maintenance of genome stability. Based on the sensitivity of mutants to genotoxic stress the SMC5/6 complex is thought to play imminent role in DNA stabilization during repair by encircling DNA at the site of lesion by bridging the heteroduplex of SMC5 and SMC6 by non SMC kleisin components NSE1, 3 and 4. In this study, we tested how formation of the SMC5/6 circular structure affects mutant sensitivity to genotoxic stress, kinetics of DSB repair and insertion mutagenesis. In the moss Physcomitrella patens SMC6 and NSE4 are essential single copy genes and this is why we used blocking of transcription to reveal their mutated phenotype. Even slight attenuation of transcription by dCas9 binding was enough to obtain stable lines with DSB repair defect and specific bleomycin sensitivity. Whereas survival after bleomycin or MMS treatment fully depends on active SMC6, NSE4 has little or negligible effect. We conclude that whereas circularization of SMC5/6 provided by the kleisin NSE4 is indispensable for the immediate NHEJ DSB repair response, other functions associated with SMC5/6 complex are critical to survive DNA damage.

Characterization of the NSE6 subunit of thePhyscomitrium patensPpSMC5/6 complex

“…Most SMC5/6 subunits are essential for the survival in most eukaryotic organisms, including plants (Diaz & Pecinka, 2018). Therefore, we first prepared P. patens mutant lines with reduced transcript levels using inactive dCas9 (Holá et al , 2021) to ensure their viability. We targeted the first exon of PpNSE6 with dCas9-sgRNA and obtained stably transformed lines.…”

“…Growth-response to double-strand break (DSB) damage induced by radiomimetic drug bleomycin was used for characterization of defects in DNA repair processes of Ppnse6dCas9_3 and Ppnse6KO1_47 mutant lines (Holá et al ., 2021). Consistently with the assumed role of SMC5/6 in DSB repair, Ppnse6dCas9_3 and Ppnse6KO1_47 were sensitive to bleomycin, with growth reduced to 55% and 35% of untreated controls, respectively (Fig.…”

“…Interestingly, we were able to generate viable Ppnse6KO lines (Fig. 4), while our attempts to prepare Ppsmc6KO lines failed (Holá et al ., 2021). Nevertheless, the Ppnse6 mutant lines exhibited severe phenotypes with reduced cell viability and high sensitivity to DNA damage, similar to other moss Ppsmc5/6 mutant lines (Holá et al ., 2021) or other plant mutants (Diaz & Pecinka, 2018).…”

The Czech Plant Nucleus Workshop 2021

“…M. Holá (IEB) presented functional characterization of SMC5/6 in P. patens. Sensitivity assays revealed a critical role of SMC5/6 in double-strand-break (DSB) repair and proposed that the circularization of SMC5/6 complex by the kleisin subunit NSE4 is indispensable for the P. patens SMC5/6 function (Holá et al 2021).…”

Molecular biology of mosses

“…Further, Trogu et al reported the results of a multiplex gene editing technique for seven PHY genes using Multiplex CRISPR-Cas9 mutagenesis (Trogu et al 2020), and Furumizu and Sawa reported the molecular evolution of the subfamily X of Leucine-rich repeat receptor-like kinases using genomic information from a broad range of land plants, including hornworts, mosses and a liverwort (Furumizu and Sawa 2021). Studies on abiotic or biotic stress responses in P. patens include the functional analysis of the structural maintenance of chromosome protein complex, SMC5/6 complex, on DNA double strand break repair caused by genotoxic stress (Holá et al 2021), the conserved and species-specific plant defense mechanisms to a fungal pathogen using a transcriptomic approach (Reboledo et al 2021), and the comprehensive identification of small secreted peptides, some of which participate in immune responses such as to chitosan treatment (Lyapina et al 2021). Ríos-Meléndez reported the results of physiological and transcriptomic analyses of the moss Pseudocorssidium replicatum, which has high desiccation tolerance (Ríos-Meléndez et al 2021).…”