4 nitrobenzyl alcohol

Single-molecule experiments were performed at room temperature (23 ± 1°C) on an objective-type total-internal-reflection fluorescence microscope (Olympus IX83 cellTIRF). Microfluidic imaging chambers were passivated with a mixture of PEG and biotin-PEG (Laysan Bio), and incubated with 0.8 µM streptavidin (Invitrogen) followed by 2 nM fluorescently labeled, His-tagged bMRP1 that had been preincubated with biotinylated anti-His₆ antibodies (Invitrogen) for 1 hr on ice in a buffer containing 50 mM Tris-HCl pH 8.0, 150 mM KCl, 2 mM MgCl₂, 0.06% digitonin, 0.5 mg/mL BSA, 10 mM phosphocreatine (Sigma), and 0.1 mg/mL creatine kinase (Sigma). A triplet-state quenching cocktail (Dave et al., 2009 (link)) of 1 mM cyclooctatetraene (Sigma), 1 mM 4-nitrobenzyl alcohol (Sigma), and 1 mM Trolox (Sigma), as well as an oxygen scavenging system (Aitken et al., 2008 (link)) containing 10 nM protocatechuate-3,4-dioxygenase (Sigma) and 2.5 mM protocatechuic acid (Sigma) were supplemented to the imaging buffer. ATP and/or LTC₄ were included in the imaging buffer at concentrations specified in the text. Fluorescence signals were split with a W-View Gemini-2C (Hamamatsu), directed to two CMOS cameras (Flash 4.0 v3, Hamamatsu), and acquired by MetaMorph software (Molecular Devices) at a frame rate specified in the text.

Optical traps tethering a single bare DNA or nucleosomal DNA were moved to a separate channel containing 2 nM Cy3- or LD650-ORC and 5 nM Cdc6 in an imaging buffer containing 25 mM Tris-OAc pH 7.5, 5% glycerol, 40 µg/ml BSA, 3 mM DTT, 2 mM TCEP, 0.1 mM EDTA, 10 mM Mg(OAc)₂, 50 mM potassium glutamate, and 2.5 mM ATP. The imaging buffer was supplemented with an ATP-regeneration system [60 mg/ml creatine phosphokinase (Sigma) and 20 mM phosphocreatine (Sigma)], a triplet-state quenching cocktail [1 mM cyclooctatetraene (Sigma), 1 mM 4-nitrobenzyl alcohol (Sigma) and 1 mM Trolox (Sigma)], as well as an oxygen scavenging system [10 nM protocatechuate-3,4-dioxygenase (Sigma) and 2.5 mM protocatechuic acid (Sigma)]. Kymographs were typically recorded for 4–10 min.