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  • Guacci Michaelis Rao DNA Hunt Hassold DNA


    简介粘蛋白复合体不仅将复制后增殖细胞中的姐妹染色单体结合在一起(Guacci等人,1997年,Michaelis等人,1997年),而且在间期组织染色质纤维的拓扑结构(Rao等人,2017年)。前者涉及不同DNA分子之间的相互作用,这些分子必须在很长的时间内保持稳定,对于减数分裂细胞来说可能是数年(Hunt和Hassold,2010年),后者涉及到来自同一DNA分子的序列之间的短暂的长程相互作用,这些序列将染色体DNA组织成染色单体样的线,环从一个中心轴发出(Klein等人。, 1999, Tedeschi et al., 2013). Given these differences, the actual mechanisms are likely to be different. It has been suggested that sister chromatid cohesion is mediated by co-entrapment of sister DNAs within a tripartite ring formed by pairwise interactions between cohesin’s Smc1, Smc3, and kleisin (Scc1) subunits (Haering et al., 2002) while chromatid-like structures during interphase are created by a DNA translocase associated with cohesin that progressively extrudes ever-longer loops of DNA (Nasmyth, 2001), an activity thought to be responsible for creating the topologically associated domains (TADs) observed using HiC (Fudenberg et al., 2016, Haarhuis et al., 2017, Rao et al., 2017, Sanborn et al., 2015, Schwarzer et al., 2017, Wutz et al., 2017). Whether loop extrusion also involves entrapment of DNAs within cohesin rings is not known.Understanding the detailed topology of cohesin’s interactions with DNA while it confers cohesion or undergoes loop extrusion is therefore crucial to understanding these two rather different functions. A key aspect of this topology is the potential for DNAs to be entrapped inside a variety of compartments within rings created by multiple interactions between cohesin’s Smc1, Smc3, and Scc1 subunits. Smc1 and Smc3 are rod-shaped proteins with dimerization domains at one end and ABC-like ATPase domains at the other, connected by 50-nm-long coiled coils. Dimerization creates V-shaped Smc1/Smc3 heterodimers with a hinge at their junction and ATPases at their vertices (Haering et al., 2002, Haering et al., 2004). The association of Scc1’s N- and C-terminal domains with the coiled coil emerging from Smc3's ATPase (its neck) and the o-Phenanthroline of Smc1’s ATPase, respectively, creates a huge SMC-kleisin (SK) ring (Gligoris et al., 2014, Haering et al., 2004). Additional interactions between Smc1 and Smc3 in the vicinity of their ATPase heads may divide the large ring created by joining Smc hinge and Smc/kleisin interfaces into two sub-compartments as described in the present study, namely a “SMC (S) compartment” created by the Smc1/3 hinge and Smc1/3 head interactions and a “kleisin (K) compartment” defined by Smc1/3 head interactions and interactions of each ATPase head with the N- and C-terminal domains of Scc1.Work on related Smc/kleisin and Rad50 complexes suggests that Smc heads in fact interact in two very different ways (Diebold-Durand et al.,2017年,Lammens等人,2011年,Lim等人,2011年,Minnen等人,2016年)。第一种方法涉及到结合在一个头部(Smc1)上的ATP与其伴侣(Smc3)上的签名图案(反之亦然)之间的相互作用,从而形成一个夹杂着一对ATP分子的复合体(Arumugam等人,2003年,Lammens等人,2004年,Marcos Alcalde等人,2017年)。这种ATP诱导的头部接触(E)是两个ATP分子水解的先决条件,这两个ATP分子会触发分离。来自枯草芽孢杆菌的Smc复合体通过两个ATP酶头部的旋转形成一种新的状态,这种状态将它们的两个特征基序和颈部并列(特征基序并置[J])(Diebold Durand等人。, 2017). This process may be facilitated by interactions between their coiled coils in the vicinity of a pronounced disruption known as the joint (Diebold-Durand et al., 2017). If the cohesin ring also undergoes a similar switch, which was one of the goals of the current study, then it could create five different types of compartments: S and K compartments associated with E and J heads as well as open SK rings in which neither heads nor coiled coils are juxtaposed (Figure 1A).Specific Smc/kleisin interactions have hitherto been detected in vivo using a bi-functional thiol-specific cross-linking reagent, BMOE, to induce rapid cross-linking between cysteine residue pairs inserted within individual ring interfaces (Gligoris et al., 2014). The results of these experiments imply that about 25% of cohesin rings are cross-linked simultaneously at all three Smc1/3 hinge, Smc3/Scc1, and Smc1/Scc1 interfaces (Gligoris et al., 2014). Chemical closure in this manner can then be exploited to detect DNA entrapment. Thus, entrapment of individual circular DNAs by chemically circularized cohesin rings leads to a modest retardation in their migration during gel electrophoresis even when all proteins have been denatured by heating in the presence of SDS (Haering et al., 2008). Likewise, co-entrapment of monomeric sister DNAs within chemically circularized cohesin causes them to migrate as dimers instead of monomers. Because they are catenated exclusively by cohesin rings, these sister DNA pairs are known as catenated dimers (CDs) (Gligoris et al., 2014). Analysis of numerous mutants has revealed a perfect correlation between the incidence of CDs and whether cells had established sister chromatid cohesion (Srinivasan et al., 2018). Thus, co-entrapment of sister DNAs within individual cohesin rings provides a mechanistic explanation for cohesion and for how cleavage of Scc1 by separase triggers sister chromatid disjunction at anaphase (Uhlmann et al., 2000).These studies have not hitherto taken into account the possibility that DNAs are entrapped within the ring’s sub-compartments, namely S or K compartments associated with E or J heads. Indeed, it has been proposed on numerous occasions that cohesion is in fact conferred by entrapment within E-S compartments and that the interconnection of E heads by kleisin merely reinforces this entrapment (Elbatsh et al., 2016, Huber et al., 2016, Li et al., 2017, Murayama et al., 2018, Murayama and Uhlmann,2015年,Stigler等人,2016年,Uhlmann,2009年,Uhlmann,2016年)。这一观点的支持源于以下观察结果:通过HOS1去乙酰化酶的失活而取消Smc3去乙酰化会延迟后期姐妹染色单体分离,尽管Scc1切割有效(Li等人,2017)。如果dna真的被截留在E-S室中,那么分离酶对其螺旋状螺旋的裂解将抑制延迟分离,这正是我们所发现的。在头部参与的复合体的S或K室中的包埋同样与以下主张一致:即凝聚力可以通过假定不能水解ATP的活性Smc1D1164E突变来建立(Chamdere等人。, 2015, Çamdere et al., 2018, Elbatsh et al., 2016).