Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin associationin vivo

Abstract

The Smc5/6 complex is involved in various DNA transactions and is best known for ensuring the fidelity of homologous recombination. We exploit single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association. We show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits we show that the Nse3 double-stranded DNA binding activity and the two arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the ssDNA binding activity at the hinge region does not prevent chromatin association. However, unlike a mutant attenuating chromatin association, a mutant that disrupts ssDNA binding results in highly elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.The structural maintenance of chromosomes (SMC) complexes cohesin, condensin and Smc5/6 are critical for the correct organisation of chromosome architecture 1 . Whereas the functions of cohesin and condensin are increasingly well understood, the Smc5/6 complex remains relatively ambiguous. Smc5/6 is conserved across all eukaryotes and is best known for its role in the cellular response to DNA damage by ensuring the fidelity of homologous recombination repair (HRR) 2,3 . Smc5/6 has been reported to promote replication fork stability 4 and facilitate DNA replication through natural pausing sites 5 . Biochemically, the complex can regulate prorecombinogenic helicases 6,7 . It has also been proposed to monitor DNA topology 8 and recently been shown to restrict viral transcription 9,10 . Complete inactivation of the Smc5/6 complex in a variety of organisms leads to cell death. However, hypomorphic mutants show significant defects in sister-chromatid HRR, display replication fork instability, are sensitive to a wide range of genotoxins and accumulate unresolved recombination intermediates 4,11,12 .Like all SMC complexes, the core of Smc5/6 is composed of two folded proteins, Smc5 and Smc6, which form a heterodimer ( Figure 1A). Each subunit comprises a long coiled-coil arm with a hinge region at one end and a globular ATPase head at the other 1 . All three SMC heterodimers interact at the hinge and ATP binding/hydrolysis occurs in two pockets formed between the heads of the two subunits. For all SMC complexes, ATP turnover is essential for cell viability and has been proposed to bring about conformational changes in the arms 13,14,15 .The ATPase activity is also key to the interaction of SMC's with DNA: Cohesin's ATPase is required for both loading and dissociation from DNA 16 , whilst condensin is dependent on its ATPase activity for translocating along DNA and forming loop structures 17,18 . The role of the Smc5/6 ATPase in DNA asso...

Nse5/6 is a negative regulator of the ATPase activity of the Smc5/6 complex

“…58 ) , that disrupts (but does not abolish) dsDNA binding. Furthermore, deletion of the gene encoding Nse6 results in an almost complete loss of chromatin associated Smc5/6 25 .…”

“…Nse5/6 is thought to promote recruitment to, or 'loading' of, the Smc5/6 complex onto chromatin 22,23 in a manner similar to that described for the cohesin 'loader complex' Scc2-Scc4 20 [reviewed in refs: 6,24]. In support of this hypothesis: S. pombe cells lacking Nse5/6 display a drastic reduction in the amount of Smc5/6 associated with chromatin 22,25 ; in S. cerevisiae, hypomorphic mutations of Nse5 lead to reduced levels of Smc5/6 associated with stalled replication forks 26 ; in humans the SLF1-SLF2 complex has been shown to recruit Smc5/6 to collapsed replication forks 21 .…”

“…At least part of the Smc5/6-recruitment function appears to be mediated by interaction of Nse5/6 with the multi-BRCT scaffold protein Rtt107 (Brc1 in S. pombe ) 27 , which itself is recruited to sites of DNA damage via binding of its C-terminal BRCT-pair to γH2A. Notably, in fission yeast, Brc1 is a dosage compensator for a hypomorphic mutation of Smc6 known as smc6-74 that leads to sensitivity to genotoxic agents and reduced levels of chromatin loading of Smc5/6 25,28 . Suppression is however dependent on the activity of the structure specific endonucleases Slx1-Slx4 and Mus81-Eme1 29 .…”

Nse5/6 inhibits the Smc5/6 ATPase to facilitate DNA substrate selection

“…This conformation is incompatible with the rod conformation. At the least, it opens the arms in the head-proximal area, yielding a more open ring-like complex that has been observed for cohesin by cryo-EM (Higashi et al ., 2020; Shi et al ., 2020) and characterized in Smc-ScpAB by electron paramagnetic resonance and cross-linking (Etheridge et al ., 2020; Nunez et al ., 2021). Upon ATP hydrolysis the heads disengage, the coiled-coils zip back up, and the complex reverts back into the J-state.…”

“…It also has a role in recruiting the Smc5/6 complex to DNA damage sites through an interaction between an N-terminal unstructured peptide in Nse6 and a multi-BRCT domain of Rtt107 (Leung et al ., 2011; Wan et al ., 2019). Single-molecule tracking recently suggested a function for Nse5/6 in chromosomal loading of Smc5/6 (Etheridge et al ., 2020).…”