FIG. 7.

PCNA and CAF-1 are corecruited to DNA containing single-strand breaks. (A) Schematic of an assay for factors which are recruited to magnetic bead-linked DNA containing single-strand breaks induced by DNase I. (B) Bead-linked DNA substrates containing different amounts of DNase I-induced single-strand breaks were incubated in a human cell-free system in the presence of either ATP (4 mM) or ATPγS (4 mM). Specific proteins bound to DNA were detected by Western blotting with antibodies against CAF-1 p150, CAF-1 p60, PCNA, and RF-C p140. (C) Preferential binding of slower-migrating forms of CAF-1 p60 to bead-linked DNA containing either single-strand breaks (lane 4) or UV photoproducts (lane 7). HeLa nuclear extract (lanes M) was loaded alongside the proteins bound to bead-linked DNA to reveal the multiple phosphorylated forms of CAF-1 p60 (indicated by a bracket) present in the cell extract (47, 49). The HeLa nuclear extract shown in lane 1 was preincubated with λ phosphatase (Ppase) to reveal the migration of dephosphorylated CAF-1 p60.