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. 1998 Nov 2;143(3):563–575. doi: 10.1083/jcb.143.3.563

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Titration conditions for recombinant CAF-1 to support nucleosome assembly during UV-induced synthesis and competence of various phosphorylated forms in the assay. (A) Plasmid DNA, either treated with UV-C at 500 J/m² (+) or not treated (−) were incubated in a cytosolic extract in the presence of [α-³²P] dCTP. Complementation in the supercoiling assay was achieved with increasing amounts of recombinant CAF-1 (solid triangle), 0 ng (lanes 2, 3, and 4), 5 ng (lane 5), 10 ng (lane 6), 20 ng (lane 7), or 40 ng (lane 8). Deproteinized DNA was purified and analyzed by agarose gel electrophoresis. The incorporation of radiolabel due to the UV-dependent DNA synthesis was visualized by autoradiography (Labeled DNA) and the total population of DNA molecules by ethidium bromide staining of the gel (Total DNA). Plasmids were processed as indicated in Materials and Methods. The positions of the supercoiled (form I), nicked (form Ir), and closed circular (form II) forms of plasmid DNA are indicated. Control supercoiled DNA was run in parallel (lane 1). (B) Recombinant CAF-1 was treated with Lambda phosphatase alone (lane 1), in the presence of β-glycerophosphate (lane 2) or mock treated (lanes 3 and 5), mitotic extract (M) and nuclear interphasic extract (I) were prepared as described (see Materials and Methods). All samples were processed for Western blot analysis and detection with the polyclonal anti–p60 antibody (p60). Bracket, various phosphorylation forms. (C) The three sources of recombinant CAF-1 (r.CAF-1): treated with Lambda phosphatase alone (lane 4), in the presence of β-glycerophosphate (lane 5), or mock treated (lane 3) (shown in B, left) were used in the assay as described above. The amount added per reaction was 10 ng (defined as the critical amount in A). The presence and amount of the phosphorylated p60 subunit of CAF-1 (exogenous and endogenous) at the end of each reaction was assessed by Western blotting using the polyclonal anti-p60 antibody (p60). Asterisk, slowest migrating phosphorylated form of p60.