Biology subjects

Nasmyth, K. A.

Publications and source records attributed to Nasmyth, K. A..

6 recordsLinked to original sources

bioRxiv2023

What Alphafold tells us about cohesin's retention and release from chromosomes

Cohesin is a trimeric complex containing a pair of SMC proteins (Smc1 and Smc3) whose ATPase domains at the end of long coiled coils (CC) are interconnected by Scc1. During interphase, it organizes chromosomal DNA topology by extruding loops in a manner dependent on Scc1s association with two large hook shaped proteins called SA (yeast: Scc3) and Nipbl (Scc2). The latters replacement by Pds5 recruits Wapl, which induces release from chromatin via a process requiring dissociation of Scc1s N-terminal domain (NTD) from Smc3. If blocked by Esco (Eco)-mediated Smc3 acetylation, cohesin containing Pds5 merely maintains pre-existing loops, but a third fate occurs during DNA replication, when Pds5-containing cohesin associates with Sororin and forms structures that hold sister DNAs together. How Wapl induces and Sororin blocks release has hitherto remained mysterious. In the twenty years since their discovery, not a single testable hypothesis has been proposed as to their role. Here, AlphaFold 2 (AF) three-dimensional protein structure predictions lead us to propose formation of a quarternary complex between Wapl, SA, Pds5, and Scc1s NTD, in which the latter is juxtaposed with (and subsequently sequestered by) a highly conserved cleft within Wapls C-terminal domain (CTD). AF also reveals how Scc1s dissociation from Smc3 arises from a distortion of Smc3s CC induced by engagement of SMC ATPase domains, how Esco acetyl transferases are recruited to Smc3 by Pds5, and how Sororin prevents release by binding to the Smc3/Scc1 interface. Our hypotheses explain the phenotypes of numerous existing mutations and are highly testable.

biochemistry↗

bioRxiv2022

Conformational dynamics of cohesin/Scc2 loading complex are regulated by Smc3 acetylation and ATP binding

The ring-shaped cohesin complex is the key player in sister chromatid cohesion, DNA repair, and gene transcription. The loading of cohesin to chromosomes requires the loader Scc2 and is regulated by ATP. This process is also hindered by Smc3 acetylation. However, the molecular mechanism underlying this inhibition remains mysterious. Here we identify a novel configuration of Scc2 with pre-engaged cohesin and reveal dynamic conformations of the cohesin/Scc2 complex in the loading reaction. We demonstrate that Smc3 acetylation blocks the association of Scc2 with pre-engaged cohesin by impairing the interaction of Scc2 with Smc3s head. Lastly, we show that ATP binding induces the cohesin/Scc2 complex to clamp DNA by promoting the interaction between Scc2 and Smc3 coiled coil. Our results illuminate a dynamic reconfiguration of the cohesin/Scc2 complex during loading and indicate how Smc3 acetylation and ATP regulate this process.

biochemistry↗

bioRxiv2022

Sister chromatid cohesion establishment during DNA replication termination

The cohesin complex tethers sister chromatids together from the moment they are generated in S-phase until their separation in anaphase1,2. This fundamental phenomenon, called sister chromatid cohesion, underpins orderly chromosome segregation. The replisome complex coordinates cohesion establishment with replication of parental DNA3. Cohesion can be established by cohesin complexes bound to DNA before replication4,5, but how replisome interaction with pre-loaded cohesin complexes results in cohesion is not known. Prevailing models suggest cohesion is established by replisome passage through the cohesin ring or by transfer of cohesin behind the replication fork by replisome components5. Unexpectedly, by visualising single replication forks colliding with pre-loaded cohesin complexes, we find that cohesin is pushed by the replisome to where a converging replisome is met. Whilst the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA. We demonstrate that these cohesin molecules tether the newly replicated sister DNAs together. Our results support a new model where sister chromatid cohesion is established during DNA replication termination, providing important insight into the molecular mechanism of cohesion establishment.

biophysics↗

bioRxiv2022

Formation of the clamped state by Scc2 and DNA overcomes the constraints imposed by zipping-up of the SMC coiled coils on cohesin's ATPase

In addition to mediating sister chromatid cohesion, cohesin, by virtue of Loop Extrusion (LE), organises the spatial arrangement of interphase DNA. The latter activity relies on DNA and Scc2 dependent ATP hydrolysis by cohesin. How the impetus from the ATPase cycle translates into reeling of DNA loops into the SMC kleisin rings is still unclear. The SMC coiled coils show several striking structural features like folding and zipping-up, if and how these structural states affect cohesins activity is still unclear. We show here that cohesins loop extruding motor contains an internal constraint that regulates its ATPase activity, zipping-up of the coiled coils impedes ATP hydrolysis by cohesin. We show that integrity of a region where the coiled coils emerge for the SMC hinge domains, SMC wrist, is critical for the zipping up of the coiled coils and the resulting inhibition of cohesins ATPase. Clamping of DNA by Scc2 onto the engaged SMC heads in the presence of ATP leads to unzipping of the coiled coils and permits ATP hydrolysis. Strikingly, irreversible folding of the coiled coils at the elbow region does not lead to any measurable change to the ATPase activity suggesting that recurrent cycles of folding and unfolding of the coiled coils is not necessary for driving continuous ATP hydrolysis by cohesin.

biochemistry↗

bioRxiv2022

DNA passes through cohesin's hinge as well as its Smc3-kleisin interface

The ring model (Haering et al. 2002) proposes that sister chromatid cohesion is mediated by co-entrapment of sister DNAs inside a tripartite cohesin ring created by a pair of rod-shaped proteins (Smc1 and Smc3) whose two ends are connected through dimerization of their hinges at one end and by association of their ATPase domains at the other end with the N- and C-terminal domains of a kleisin subunit (Scc1). The model explains how Scc1 cleavage triggers anaphase (Uhlmann, Lottspeich, and Nasmyth 1999) but has hitherto only been rigorously tested using small circular mini-chromosomes in yeast, where crosslinking the rings three interfaces, creating a covalent circular molecule, induces catenation of individual sister DNAs (Haering et al. 2008; Srinivasan et al. 2018). If the model applies to real chromatids, then the ring must have a DNA entry gate essential for mitosis. Whether this is situated at the Smc3/Scc1 (Murayama and Uhlmann 2015; Murayama et al. 2018) or Smc1/Smc3 hinge (Gruber et al. 2006) interface is an open question. Using an in vitro system (Collier et al. 2020), we show that cohesin in fact possesses two DNA gates, one at the Smc3/Scc1 interface and a second at the Smc1/3 hinge. Unlike the Smc3/Scc1 interface, passage of DNAs through SMC hinges depends on both Scc2 and Scc3, a pair of regulatory subunits necessary for entrapment in vivo (Srinivasan et al. 2018). This property together with the lethality caused by locking this interface but not that between Smc3 and Scc1 in vivo (Gruber et al. 2006) suggests that passage of DNAs through the hinge is essential for building sister chromatid cohesion. Passage of DNAs through the Smc3/Scc1 interface is necessary for cohesins separase-independent release from chromosomes (Chan et al. 2012) and may therefore largely serve as an exit gate.

biochemistry↗

bioRxiv2019

Scc2 counteracts a Wapl-independent mechanism that releases cohesin from chromosomes during G1 but is unnecessary during S phase for establishing cohesion

Cohesins association with chromosomes is determined by loading dependent on the Scc2/4 complex and release due to Wapl. We show here that Scc2/4 is not merely a loading complex and that it actively maintains cohesin on chromosomes during G1. It does so by blocking a Wapl-independent release reaction that requires opening the cohesin ring at its Smc3/Scc1 interface as well as the D loop of Smc1s ATPase. The Wapl-independent release mechanism is switched off as cells activate Cdk1 and enter G2/M and cannot be turned back on without cohesins dissociation from chromosomes. The latter phenomenon enabled us to show that cohesin that has already captured DNA in a Scc2-dependent manner before replication no longer requires Scc2 to capture sister DNAs during S phase.

molecular biology↗