The Smc5/6 Core Complex Is a Structure-Specific DNA Binding and Compacting Machine

Serrano, Diego; Cordero, Gustavo; Kawamura, Ryo; Sverzhinsky, Aleksandr; Sarker, Muzaddid; Roy, Shamayita; Malo, Catherine; Pascal, John M.; Marko, John F.; D’Amours, Damien

doi:10.1016/j.molcel.2020.11.011

Cited by 55 publications

(71 citation statements)

References 63 publications

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“…The possible existence of elbows in the arms of yeast Smc5/6 was previously reported, but not without controversy. Here, we found no convincing evidence for the folding of arms in yeast Smc5/6, with similar conclusions recently being drawn for the same complex (Yu et al , 2021) and a human Smc5/6 core complex (Serrano et al , 2020). While some XL‐MS inter‐domain cross‐links are consistent with a folded state, these cross‐links imply different folding points, more often than not being inconsistent with the predicted discontinuities in the heptad repeat of the Smc5/6 coiled coils (Burmann et al , 2019), presumed to support the arm folding (found one third of the way from the hinge).…”

Section: Discussionsupporting

confidence: 83%

“… Spotting assay to determine sensitivity of yeast strains to UV irradiation and treatment with methyl methanesulphonate (MMS) or hydroxyurea (HU). A strain lacking the N‐terminal region of Nse6 ( nse6(86‐C) ) does not show increased sensitivity compared to the wild type, while a control strain ( smc6(R135E) ) recently described by others (Serrano et al , 2020 ) does. …”

Section: Resultsmentioning

confidence: 94%

“…We thus suspect that Nse5/6 (or Slf1/2) is needed for proper activity of this complex. It will be interesting to compare hexamer and octamer activity in recently reported single‐molecule assays (Gutierrez‐Escribano et al , 2020 ; Serrano et al , 2020 ). Inhibition of a SMC ATPase by an associated subunit has previously been reported for E. coli MukBEF (Zawadzka et al , 2018 ).…”

Section: Discussionmentioning

confidence: 99%

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Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

et al. 2021

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Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo‐complex. We found that the Nse5/6 sub‐complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross‐linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross‐linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non‐productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.

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Section: Discussionsupporting

confidence: 83%

Section: Resultsmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

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Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

et al. 2021

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“…More direct evidence for a role for SMC5/6 in DNA compaction comes from two very recently published in vitro studies. Single-molecule magnetic tweezer assays showed that both yeast (Gutierrez-Escribano et al, 2020) and human (Serrano et al, 2020) SMC5/6 complexes were able to compact DNA in an ATP-dependent fashion. These biophysical studies therefore support a functional role for the SMC5/6 complex in compacting chromatinized, extrachromosomal viral DNA.…”

Section: Articlementioning

confidence: 99%

“…Exactly how SLF2 recruits the SMC5/6 complex to extrachromosomal viral DNA remains unclear. The recently published in vitro DNA compaction studies showed preferential SMC5/6 binding to and compaction of DNA with unusual tertiary structures such as supercoiling (Gutierrez-Escribano et al, 2020;Serrano et al, 2020). These DNA structures may form the basis for SMC5/6 recognition of extrachromosomal viral DNA, especially given the known supercoiling of HBV cccDNA, which is similarly restricted by the SMC5/6 complex (Decorsiè re et al, 2016;Murphy et al, 2016).…”

Section: Articlementioning

confidence: 99%

The SMC5/6 complex compacts and silences unintegrated HIV-1 DNA and is antagonized by Vpr

Dupont

Bloor

Williamson

et al. 2021

Cell Host & Microbe

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Silencing of nuclear DNA is an essential feature of innate immune responses to invading pathogens. Early in infection, unintegrated lentiviral cDNA accumulates in the nucleus yet remains poorly expressed. In HIV-1like lentiviruses, the Vpr accessory protein enhances unintegrated viral DNA expression, suggesting Vpr antagonizes cellular restriction. We previously showed how Vpr remodels the host proteome, identifying multiple cellular targets. We now screen these using a targeted CRISPR-Cas9 library and identify SMC5-SMC6 complex localization factor 2 (SLF2) as the Vpr target responsible for silencing unintegrated HIV-1. SLF2 recruits the SMC5/6 complex to unintegrated lentiviruses, and depletion of SLF2, or the SMC5/6 complex, increases viral expression. ATAC-seq demonstrates that Vpr-mediated SLF2 depletion increases chromatin accessibility of unintegrated virus, suggesting that the SMC5/6 complex compacts viral chromatin to silence gene expression. This work implicates the SMC5/6 complex in nuclear immunosurveillance of extrachromosomal DNA and defines its targeting by Vpr as an evolutionarily conserved antagonism.

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Crucial role of the NSE1 RING domain in Smc5/6 stability and FANCM-independent fork progression

Lorite,

Apostolova,

Guasch-Vallés

et al. 2024

Cell. Mol. Life Sci.

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The Smc5/6 complex is a highly conserved molecular machine involved in the maintenance of genome integrity. While its functions largely depend on restraining the fork remodeling activity of Mph1 in yeast, the presence of an analogous Smc5/6-FANCM regulation in humans remains unknown. We generated human cell lines harboring mutations in the NSE1 subunit of the Smc5/6 complex. Point mutations or truncations in the RING domain of NSE1 result in drastically reduced Smc5/6 protein levels, with differential contribution of the two zinc-coordinating centers in the RING. In addition, nse1-RING mutant cells display cell growth defects, reduced replication fork rates, and increased genomic instability. Notably, our findings uncover a synthetic sick interaction between Smc5/6 and FANCM and show that Smc5/6 controls fork progression and chromosome disjunction in a FANCM-independent manner. Overall, our study demonstrates that the NSE1 RING domain plays vital roles in Smc5/6 complex stability and fork progression through pathways that are not evolutionary conserved.

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The Smc5/6 Core Complex Is a Structure-Specific DNA Binding and Compacting Machine

Cited by 55 publications

References 63 publications

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

The SMC5/6 complex compacts and silences unintegrated HIV-1 DNA and is antagonized by Vpr

Crucial role of the NSE1 RING domain in Smc5/6 stability and FANCM-independent fork progression

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