Characterization of the NSE6 subunit of the<i>Physcomitrium patens</i>PpSMC5/6 complex

Lelkes, Edit; Holá, Marcela; Jemelkova, Jitka; Štefanovie, Barbora; Kolesar, Peter; Vágnerová, Radka; Adamus, Marek; Angelis, Karel J.; Paleček, Jan

doi:10.1101/2022.07.28.501545

Cited by 3 publications

(30 citation statements)

References 63 publications

(103 reference statements)

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“…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 (Fig. 1D; (Lelkes et al , 2023)). The Stag-PpNSE6(aa1-370) specifically precipitated PpNSE5(aa190-526), while not precipitating PpNSE5(aa245-526) (Fig.…”

Section: Resultsmentioning

confidence: 86%

“…1A) and other PpNSE5 fragments (Fig. 2) against PpSMC5 and PpSMC6 constructs (Lelkes et al , 2023). Surprisingly, only PpNSE5(aa1-230) fragment bound PpSMC5 and PpSMC6 (Fig.…”

Section: Resultsmentioning

confidence: 94%

“…To analyse PpNSE5-PpSMC6 interaction further, we prepared different arm fragments of PpSMC6 truncated from the head-or hinge-proximal end (Fig. 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) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Like with PpSMC6, we wanted to delineate the PpNSE5-binding region of PpSMC5 and used a similarly designed panel of constructs (Fig. S3A; (Lelkes et al , 2023)). We found that the Gal4BD-PpSMC5(aa280-790) headless fragment interacts with both Gal4AD-PpNSE5 and Gal4AD-PpNSE6 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For the pull-down assays, pET-Duet-PpNSE6(aa1-370)-StrepTwin-Stag and pET-28a-PpNSE5(aa1-526) plasmids with codon optimized for E. coli expression were described in (Lelkes et al , 2023). The PpNSE5 fragments aa1-230 and aa72-230 (Fig.…”

Section: Methodsmentioning

confidence: 99%

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NSE5 subunit interacts with distant regions of the SMC arms in thePhyscomitrium patensSMC5/6 complex

Vaculíková,

Holá,

Králová

et al. 2024

Preprint

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Structural Maintenance of Chromosome (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. SMC complexes form long rod-like structures, which can change to ring-like and elbow-bent conformations upon binding ATP, DNA and other regulatory factors. These SMC dynamic conformational changes are involved in their loading, translocation, and DNA loop extrusion. Here, we examined the binding and role of the PpNSE5 regulatory factor of Physcomitrium patens PpSMC5/6 complex. We found that the PpNSE5 C-terminal half (aa230-505) is required for binding to its PpNSE6 partner, while the N-terminal half (aa1-230) binds PpSMC subunits. Specifically, the first 71 amino acids of PpNSE5 were required for binding to PpSMC6. Interestingly, the PpNSE5 binding required the PpSMC6 head-proximal joint region and PpSMC5 hinge-proximal arm, suggesting a long distance between binding sites on PpSMC5 and PpSMC6 arms. Given the long distance between these PpSMC sites and the size of PpNSE5, we hypothesize that PpNSE5 either links two antiparallel SMC5/6 complexes or binds one SMC5/6 in elbow-bent conformation. In addition, we generated the P. patens mutant lines with CRISPR/Cas9-integrated stop codons in PpNSE5. The mutant line (Ppnse5KO1) with an N-terminally truncated version of PpNSE5 (starting from an alternative aaMet72) exhibited DNA repair defects while keeping a normal number of rDNA repeats. As the first 71 amino acids of PpNSE5 are required for PpSMC6 binding, our results suggest the specific role of PpNSE5-PpSMC6 interaction in DNA repair. Altogether, our study suggests that PpNSE5 binding to distant regions of the PpSMC5 and PpSMC6 arms serves a specific role in loading at DNA lesions.

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Section: Resultsmentioning

confidence: 86%

“…1A) and other PpNSE5 fragments (Fig. 2) against PpSMC5 and PpSMC6 constructs (Lelkes et al , 2023). Surprisingly, only PpNSE5(aa1-230) fragment bound PpSMC5 and PpSMC6 (Fig.…”

Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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NSE5 subunit interacts with distant regions of the SMC arms in thePhyscomitrium patensSMC5/6 complex

Vaculíková,

Holá,

Králová

et al. 2024

Preprint

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The phenotype of SMC6_G514R hinge mutant ofPhyscomitrium patents

Angelis,

Holá,

Vágnerová

et al. 2024

Preprint

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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 inSchizosaccharomyces pombewithin a conserved motif. Using CRISPR/Cas9 directed oligonucleotide replacement, we have introduced this G to R point mutation in SMC6 ofPhyscomitrium 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 establishedPpsmc6_G514RandPpsmc6_G517Rlines.SinceP. patensmutants with entire or partial deletion of theSMC6gene 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. ThePpsmc6_G514Rline has aberrant morphology quite similar toPpsmc6_dCas, though the absence of protonemata branching and formation of gametophores is incomplete. On the contrary, thePpsmc6_G517Rline is morphologically more or less similar to WT. Spontaneous and bleomycin-induced mutagenesis and maintenance of the number of rDNA copies in thePpsmc6_G514Rline is also similar toPpsmc6_dCas, whilePpsmc6_G517Rmore or less mimics WT. The sensitivity of thePpsmc6_G514Rline to bleomycin is not as severe as that ofPpsmc6_dCas, and surprisingly, thePpsmc6_G517Rline 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 ofPpsmc6_G517RandPpsmc6_dCaswhen compared to WT is only slightly or moderately reduced, it is completely abolished inPpsmc6_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 inPpsmc6_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.

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

Vaculíková,

Holá,

Králová

et al. 2024

The Plant Journal

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SUMMARYStructural maintenance of chromosome (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. SMC complexes form long rod‐like structures, which can change to ring‐like and elbow‐bent conformations upon binding ATP, DNA, and other regulatory factors. These SMC dynamic conformational changes are involved in their loading, translocation, and DNA loop extrusion. Here, we examined the binding and role of the PpNSE5 regulatory factor of Physcomitrium patens PpSMC5/6 complex. We found that the PpNSE5 C‐terminal half (aa230–505) is required for binding to its PpNSE6 partner, while the N‐terminal half (aa1–230) binds PpSMC subunits. Specifically, the first 71 amino acids of PpNSE5 were required for binding to PpSMC6. Interestingly, the PpNSE5 binding required the PpSMC6 head‐proximal joint region and PpSMC5 hinge‐proximal arm, suggesting a long distance between binding sites on PpSMC5 and PpSMC6 arms. Therefore, we hypothesize that PpNSE5 either links two antiparallel SMC5/6 complexes or binds one SMC5/6 in elbow‐bent conformation, the later model being consistent with the role of NSE5/NSE6 dimer as SMC5/6 loading factor to DNA lesions. In addition, we generated the P. patens Ppnse5KO1 mutant line with an N‐terminally truncated version of PpNSE5, which exhibited DNA repair defects while keeping a normal number of rDNA repeats. As the first 71 amino acids of PpNSE5 are required for PpSMC6 binding, our results suggest the role of PpNSE5–PpSMC6 interaction in SMC5/6 loading to DNA lesions.

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Characterization of the NSE6 subunit of thePhyscomitrium patensPpSMC5/6 complex

Cited by 3 publications

References 63 publications

NSE5 subunit interacts with distant regions of the SMC arms in thePhyscomitrium patensSMC5/6 complex

NSE5 subunit interacts with distant regions of the SMC arms in thePhyscomitrium patensSMC5/6 complex

The phenotype of SMC6_G514R hinge mutant ofPhyscomitrium patents

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

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