Figure 9.
The capacity of CAF-1 to support nucleosome assembly during UV- induced synthesis depends on both the number of lesion/repair sites and the phosphorylation form of CAF-1. (A) Plasmid DNA, either treated with UV-C (150 and 300 J/m2) or not treated (0) 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, which was either not treated (solid triangle) or treated before its use with Lambda phosphatase (open triangle): 5 ng (lanes 3, 7, 11, and 15), 10 ng (lanes 4, 8, 12, and 16), 20 ng (lanes 5, 9, 13, and 17) or 40 ng (lanes 6, 10, 14, and 18). Deproteinized DNA was purified and analysed by agarose gel electrophoresis. The incorporation of radiolabel due to the UV-induced 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 (C) was run in parallel. 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. (B) Data from experiments as described above using plasmid DNA treated with UV-C (150, 300, and 500 J/m2) and increasing amounts of recombinant CAF-1 either not treated (___) or treated before its use with Lambda phosphatase (- - -) were quantified using a PhosphorImager and ImageQuant software. The percentage of form I in the labeled plasmid (%) is represented as a function of the amount of CAF-1 provided in various phosphorylation status (ng).