Despite 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.

1-Acyl-7-nitroindolines have been found to be useful photoactivated protecting groups for rapid release of carboxylates in aqueous solution. Mechanistic details of carboxylic acid photorelease from model compounds in solutions of varying H₂O–CH₃CN composition are now reported, using data from product studies, deuterium isotope effects, kinetic studies (UV-Vis), and nanosecond laser flash photolysis. Our data support a mechanism (via T₁) that involves reaction of a critical photogenerated intermediate 21 (an acetic nitronic anhydride), obtained via photochemical transfer of the acetyl group from the amide nitrogen to one of the oxygen atoms of the nitro group. This mode of transfer has been reported in the photochemistry of a variety of N-acetyl-o-nitrodiphenylamines. Two competing pathways of reaction from 21 that differ in how the acyl group is cleaved are proposed to account for the products observed. In solutions of higher water content, the predominant reaction pathway of 21 is via an A_AL1-like cleavage that results in formal intramolecular redox reaction of the aromatic ring system, to give the released carboxylic acid and 7-nitrosoindole 3 (after tautomerisation of an initially formed nitroso-3H-indole 9). In solutions of low water content, the major pathway for hydrolysis of 21 is via the standard addition–elimination mechanism (A_AC2) with water as the nucleophile, that releases the carboxylic acid and nitroindoline 4. Laser flash photolysis studies in wholly aqueous medium gave a transient (within the 20 ns laser pulse) at λ_max 450 nm, assignable to 21. Single exponential decay of this species in water (k_obs = 5 × 10⁶ s⁻¹) is assigned to the release of the carboxylic acid and formation of the nitroso-3H-indole 9, which is supported by time-resolved measurements of acid release using bromothymol blue. Therefore, 1-acyl-7-nitroindolines photorelease their protected functionalities at rates in the submicrosecond time scale.