Extensive DNA synthesis associated with repair of a site-specific single-strand DNA break. (A) Synthetic 24-mer oligonucleotides (nonmodified or containing a 1,3-intrastrand cisplatin-cross-link) were annealed to single-stranded M13 DNA and elongated by T4 DNA polymerase in the presence of dNTPs. Con and Pt DNA substrates were covalently closed with T4 DNA ligase. The omission of T4 DNA ligase resulted in the formation of circular DNA molecules containing a site-specific single-strand DNA break (Nick). The synthesis of Pt and Con DNA substrates in vitro results in a range of topoisomers distributed around the most relaxed molecules (lanes 1 and 2). This was confirmed by agarose gel electrophoresis in the presence of either chloroquine or ethidium bromide (data not shown). In contrast, nick DNA consists of form II and runs as a single band (lane 3). (B) Con, Pt, and nick DNA substrates were incubated in a Drosophila cell-free system, and site-specific DNA synthesis was localized by restriction enzyme digestion of each DNA substrate. A schematic representation of the restriction fragments flanking the lesion site is shown at the top, and the sizes (in base pairs) of labeled restriction fragments are indicated alongside each gel. Incubation times were 1 min (lanes 1, 8, and 15), 5 min (lanes 2, 9, and 16), 15 min (lanes 3, 10, and 17), 45 min (lanes 4, 11, and 18), 90 min (lanes 5, 12, and 19), 180 min (lanes 6, 13, and 20), and 360 min (lanes 7, 14, and 21). (C) Con and nick DNA substrates were incubated for 6 h with either cytosolic extract derived from human cells (with [+] or without [−] recombinant CAF-1 complex) or Xenopus egg extract at 37 or 23°C, respectively.

Single-strand gaps trigger chromatin assembly in the absence of extensive DNA synthesis or ligation. Gapped circular DNA substrates were prepared from nick DNA by exploiting the 3′-to-5′ exonuclease activity of T4 DNA polymerase in the absence of dNTPs. Con DNA was treated in parallel as a control (designated Con*). Gapped circular, nick, and Con DNA substrates were incubated in the Drosophila cell-free system for 6 h in the presence or absence of aphidicolin and then subjected to MNase digestion analysis. Two different MNase digests are shown for each reaction. The oligonucleosomal DNA bands corresponding to mono-, di-, tri-, and tetranucleosomal DNA are indicated. A 123-bp ladder was used as a molecular weight marker (M).

The amino terminus of CAF-1 p150 interacts directly with PCNA. (A) Scheme of GST fusion proteins. (B) Pull-down assay from HeLa nuclear extract using GST-p150(620-938), GST-p150(1-649), and GST-PCNA (Xenopus open reading frame). GST alone was used as a control. Input (I), unbound (U), and bound (B) fractions of proteins were revealed by Western blotting with monoclonal antibodies against PCNA and the p150 subunit of CAF-1. Twice the amount of input fraction was loaded compared to the bound and unbound fractions. (C) Pull-down of recombinant human PCNA by GST-p150(1-649), GST-p150(1-244), GST-p150(32-649), and GST-p150(32-244). GST alone was used as a control. PCNA present in the input (I), unbound (U), bound (B), 0.6 M KCl elution (E), and 0.6 M KCl-resistant (B*) fractions was revealed by Western blotting with a specific monoclonal antibody (PC10). Equal amounts of each fraction were loaded.

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.

Model for bidirectional propagation of nucleosomal arrays facilitated by the interaction of CAF-1 with PCNA sliding clamps during DNA damage processing. The initiation of this nucleosome assembly process is dependent on the presence of single-strand breaks and gaps produced either by direct DNA damage or during excision repair. The recruitment to repair sites of PCNA and the histone chaperone CAF-1 (together with H3/H4) requires ATP. The amino terminus of CAF-1 p150 is engaged in a stable interaction with specific sites on the outer front side of PCNA. The ability of the PCNA toroidal ring to slide along the DNA helix provides a possible mechanism for nucleosomal arrays to propagate bidirectionally from sites of repair (indicated by the arrows). The hatched nucleosome represents an undefined structure at the repair site.

Single-strand breaks trigger chromatin assembly in the absence of extensive DNA synthesis. (A) Supercoiling analysis of chromatin assembly on DNA substrates containing site-specific single-strand breaks after incubation in a Drosophila cell-free system. Incubation times were 1 min (lanes 2 and 10), 5 min (lanes 3 and 11), 15 min (lanes 4 and 12), 45 min (lanes 5 and 13), 90 min (lanes 6 and 14), 180 min (lanes 7 and 15), and 360 min (lanes 8 and 16). Lanes 1 and 9 contain untreated Con and nick DNA, respectively. The migration of relaxed/nicked circular DNA (Ir/II), linear DNA (III), supercoiled DNA (I), and labeled gapped circular molecules is indicated. (B) Supercoiling analysis of chromatin assembly using DNA substrates containing site-specific DNA lesions after incubation in a Drosophila cell-free system for 6 h in the presence or absence of aphidicolin (590 μM). The graph shows the quantification of the number of extensively supercoiled topoisomers (I) relative to the total topoisomer population as a percentage. The migration of relaxed/nicked circular DNA (Ir/II), linear DNA (III), supercoiled DNA (I), and labeled gapped circular molecules is indicated. (C) MNase digestion analysis of nucleosomal arrays formed on nick DNA after 6 h incubation in a Drosophila cell-free system. Two digestions with increasing amounts of MNase are shown for each reaction. The regularly spaced DNA bands corresponding to mono-, di-, tri-, and tetranucleosomal DNA are indicated. A 123-bp ladder was used as a molecular weight marker (M). (D) Effect of aphidicolin (590 μM) on nucleosomal arrays formed using nick DNA in a 6-h reaction with the Drosophila cell-free system (MNase digestion analysis was performed under conditions identical to those used for panel C). (E) Formation of nucleosomal arrays using nick DNA in a 6-h reaction with the Drosophila cell-free system in the presence of increasing amounts of aphidicolin. The distinct pattern of MNase digestion products seen in the presence of aphidicolin on labeled Nick DNA molecules in panels D and E is indicated by dashed lines. This pattern is thought to result from the assembly of nucleosomal arrays adjacent to gaps in the damaged DNA strand.

CAF-1 stimulates nucleosome assembly during single-strand break repair. For kinetic analysis of supercoiling during single-strand break repair in a human cell-free system, nick and Con DNA substrates were incubated with HeLa cytosolic extract which contains only trace amounts of endogenous CAF-1 either in the absence or in the presence of exogenous purified CAF-1 complex (1 ng/μl). Incubation times were 1 min (lanes 1 and 6), 5 min (lanes 2 and 7), 15 min (lanes 3, 8, 11, and 14), 45 min (lanes 4, 9, 12, and 15), and 180 min (lanes 5, 10, 13, and 16). The migration of relaxed/nicked circular DNA (Ir/II) and supercoiled DNA (I) molecules is indicated.

CAF-1 interacts with specific sites on the outer front side of PCNA. (A) Front and side views of the homotrimeric PCNA toroid showing positions of the mutations used in the GST pull-down assay. (B) GST pull-down of wild-type and mutant recombinant human PCNAs by GST-p150(1-649). Equivalent amounts of C-terminally histidine-tagged wild-type (WT-His) and mutant (LAPK251, QLGI125, SHV43, and VDK188; created via alanine substitutions) PCNA proteins were used in each experiment. Input (I), unbound (U), bound (B), 0.6 M KCl elution (E), and 0.6 M KCl-resistant (B*) fractions of PCNA proteins were revealed by Western blotting with a monoclonal antibody against PCNA.

PCNA mediates chromatin assembly linked to single-strand break repair. (A) Analysis of supercoiling during single-strand break repair in the Drosophila cell-free system. Supercoiled circular plasmid DNA (lane 1) was treated with DNase I to induce single-strand breaks (lane 2). These DNA substrates were incubated for 3 h at 23°C with either nondepleted extract (lanes 3 and 4), extract depleted with GST alone (lanes 5 and 6), or extract depleted with GST-p150(1-244) (lanes 7 to 10). Recombinant human PCNA was added to the reactions in lanes 9 and 10 to a final concentration of 8 ng/μl. The migration of relaxed/nicked circular DNA (Ir/II) and supercoiled DNA (I) molecules is indicated. (B) Bead-linked DNA substrates containing DNase I-induced single-strand breaks or undamaged control molecules were incubated in the Drosophila cell-free system as described for panel A for 1 min at 23°C. Bead-linked DNA substrates were equilibrated and washed in a slightly higher ionic strength buffer (40 mM HEPES-KOH [pH 7.8], 100 mM KCl, 0.05% NP-40) for reactions with the Drosophila cell-free system. PCNA binding to the bead-linked DNA substrates was detected by Western blotting. The recombinant human PCNA containing a C-terminal histidine tag migrates more slowly than endogenous Drosophila PCNA.