FACT has moderate nucleosome assembly activity. FACT has moderate nucleosome assembly activity. (A) FACT has moderate tetrasome assembly activity. 147-bp 601 DNA (30 nM) was incubated with FACT (15–240 nM) pre-equilibrated with 30 nM X. laevis or H. sapiens (H3–H4)2, separated by PAGE, and visualized by SYBR Gold (DNA). Lane 2 shows a salt-assembled tetrasome on the same DNA (Tetra). (B) FACT facilitates the deposition of H2A–H2B dimers onto the tetrasome, resulting in hexasome and nucleosome assembly. 60 nM salt-reconstituted tetrasome (lane 2) and 30 nM Atto 647N–labeled H2A–H2B were incubated with increasing amounts of FACT (10–1,280 nM), and gels were visualized by H2B fluorescence. (C) FACT facilitates the deposition of H2A–H2B onto the hexasome. 20 nM under-assembled nucleosome with fluorescence labels on H4 (Alexa 488) and H2B (Atto 647N) (lane 3) was incubated with increasing amounts of FACT, and the gel was visualized as indicated. (D) FACT (100–3,200 nM) promotes hexasome and nucleosome assembly from free histones (50 nM H3–H4; 100 nM H2A–H2B) and DNA (25 nM). FACT (at the indicated concentrations) was incubated with histones at RT for 10 min and then DNA was added, and products were visualized as indicated. The two intermediate complexes are indicated by arrows. Tao Wang et al. LSA 2018;1:e201800107 © 2018 Wang et al.