Fig. 1. Human Asf1 acts synergistically with CAF-1 to assemble nucleosomes during NER. ( A ) UV-damaged DNA substrate was incubated in a human cell-free system alone or complemented with core histones, CAF-1 (16 ng) or nuclear extract (2.5 µg protein). To these reactions, Asf1a or Asf1b (60 ng +, 250 ng + ) was added. Lane 1 contains untreated UV-damaged DNA. Radiolabel incorporation due to repair synthesis was visualized by autoradiography (labeled DNA) and total DNA by ethidium bromide staining (total DNA). Relaxed/nicked circular DNA (Ir/II) and supercoiled DNA (I) are indicated. ( B ) Analysis by MNase digestion of nucleosomal arrays formed on repaired DNA (labeled DNA) in a human cell-free system alone or complemented with histones, CAF-1 (16 ng +, 48 ng + ), Asf1a or Asf1b (60 ng) or nuclear extract as control (7.5 µg protein). Three digestion times (45 s and 3 and 6 min) are shown for each reaction. Positions corresponding to mono-, di-, tri- and tetra-nucleosomal DNA are indicated. Nuclear extract produced larger MNase digestion products, suggesting the involvement of additional factors in this extract. ( C ) Reactions as in (A) but to which Asf1a, Asf1b (60 ng) or nucleoplasmin (60 ng and 120 ng) was added.