Abstract
SummaryDespite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.
Highlights
The structural maintenance of chromosomes (SMC) protein family is conserved from prokaryotic to eukaryotic cells, and their role in DNA organization is vital for many aspects of chromosome function (Hirano, 2016; Jeppsson et al, 2014; Uhlmann, 2016)
We used a tetramer complex consisting of Psm1, Psm3, Rad21, and Psc3 (Murayama and Uhlmann, 2014), including C-terminal SNAP and CLIP tags on Psm1 and Psm3, respectively, that could be labeled with Dy547 and Alexa 647 fluorophores as a fluorescence resonance energy transfer (FRET) pair (Figures 1A and S1A)
When we superimpose the engaged DNA-bound SMC heads in our structure with the equivalent domains of Rad50 from the Rad50-Mre11-DNA complex, we find a striking overlap between the ATP-bound ATPase heads as well as the DNA (Figure S3A; Liu et al, 2016; Schu€ler and Sjogren, 2016)
Summary
The structural maintenance of chromosomes (SMC) protein family is conserved from prokaryotic to eukaryotic cells, and their role in DNA organization is vital for many aspects of chromosome function (Hirano, 2016; Jeppsson et al, 2014; Uhlmann, 2016). Among the SMC complexes, cohesin establishes cohesion between replicated sister chromatids, which forms the basis for faithful chromosome segregation during cell division. Additional roles of cohesin include chromatin domain organization in interphase as well as DNA repair by homologous recombination. Cohesin has been seen to extrude DNA loops without a need for the ring to topologically trap DNA (Davidson et al, 2019; Kim et al, 2019). Such a loop extrusion mechanism has been proposed to underlie interphase chromatin domain organization. The molecular mechanisms by which cohesin topologically entraps DNA or extrudes a DNA loop are not yet understood
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