Abstract
Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.
Highlights
The topological entrapment of chromosomes by cohesin (Smc[1], Smc[3], Scc[1], and Scc3) is central to genome integrity[1,2,3]
We present the crystal structures of Xenopus laevis Eco[2] ACT domain bound to two different substrate peptides, which reveal that Eco[1] substrate specificity is determined by the concerted conformational changes of the conserved central βhairpin and the C-terminal extension (C extension)
As a result of the enzymatic and chemical reactions, K112 and K113 were either acetylated or di-methylated at all time points. This strategy facilitated our analysis since K112 and K113 are blocked for trypsin proteolysis and the peptide TVGLKKDDYQLNDR could be consistently extracted from the gel prior to mass spectrometry analysis
Summary
The topological entrapment of chromosomes by cohesin (Smc[1], Smc[3], Scc[1], and Scc3) is central to genome integrity[1,2,3]. Cohesin is loaded onto chromatin by the Scc2-Scc[4] loader complex[4,5,6], persistent cohesion cannot be established without the acetylation of two conserved tandem lysines on the Smc[3] subunit by the Eco[1] acetyltransferase (ACT)[7,8,9,10]. Break-induced phosphorylation of Scc[1] is thought to trigger its Eco1-mediated acetylation, which antagonizes Wapl releasing activity[25]. Despite the importance of acetylation in cohesion establishment and human diseases, little is known about how Eco[1] targets its canonical substrate Smc[3] and how this acetylation stabilizes cohesin on DNA. By aligning the sequences of different acetylated cohesin peptides, we show that the target lysine of acetylation is crick.ac.uk) www.nature.com/scientificreports/
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