Volume 28, Issue 1, Pages 147-158 (October 2007) Nucleotide Excision Repair and Homologous Recombination Systems Commit Differentially to the Repair of DNA-Protein Crosslinks  Toshiaki Nakano, Soh Morishita, Atsushi Katafuchi, Mayumi Matsubara, Yusuke Horikawa, Hiroaki Terato, Amir M.H. Salem, Shunsuke Izumi, Seung Pil Pack, Keisuke Makino, Hiroshi Ide  Molecular Cell  Volume 28, Issue 1, Pages 147-158 (October 2007) DOI: 10.1016/j.molcel.2007.07.029 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 NER and HR Are Differentially Involved in the Repair of FA- and azaC-Induced DPCs (A and B) Cell survival of WT and repair-deficient cells treated with FA (A) and azaC (B). Mutants are deficient in NER (uvrA), HR (recA), translesion synthesis (umuDC), and NER+HR (uvrA recA). For strains, see Table S1. Note that the concentration of azaC is displayed on a logarithmic scale. Data points are means of two or three independent experiments. Molecular Cell 2007 28, 147-158DOI: (10.1016/j.molcel.2007.07.029) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 NER Promotes the Restoration of DPC-free DNA In Vivo (A) Characterization of chromosomal DNA isolated from FA-treated cells. After FA (10 mM) or mock treatment of WT cells, DNA in lysed cells was sheared and isolated. DNA (3 μg) was treated with the indicated amounts of Proteinase K or heat (65°C for 6 hr), embedded in the agarose plug, separated by agarose gel electrophoresis, and stained by EtBr. The leftmost lane shows size markers (23.1, 9.4, and 6.6 kbp). (B) Upper panel, formation of DPC-free DNA during postrepair incubation. Cells were exposed to 10 mM FA, and DNA was isolated after the indicated time of repair incubation. DNA (3 μg) was embedded in the agarose plug, separated by agarose gel electrophoresis, and visualized by EtBr. Lower panel, absence of CLPs in DNA fractions eluted from agarose plugs. DNA isolated as in the upper panel was labeled with FITC, separated, and visualized by fluorescence. The image was overexposed to reveal faint bands if any. The lanes correspond to those in the upper panel. (C) Formation of DPC-free DNA is independent of DNA replication. The dnaE486 mutant was preincubated at permissive (30°C) and nonpermissive (42°C) temperatures, then treated with 10 mM FA and subjected to repair incubation at the respective temperatures for the indicated times. DNA was analyzed as in the upper panel of (B). Molecular Cell 2007 28, 147-158DOI: (10.1016/j.molcel.2007.07.029) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Excision of DPCs by UvrABC Is Dependent on the Size of CLPs (A) Sequence of 60OXA-DPC. Proteins were covalently bonded to oxanine (O), and the 32P label (∗p) was introduced to the 5′ end or 5′and 3′ ends. Arrows indicate the incision sites by UvrABC. (B) PAGE analysis of the 5′ incision of 60OXA-DPCs. 60OXA-DPCs (5′ end labeled) were incubated with UvrABC for 0, 15, and 30 min. After incubation, samples were treated with Proteinase K and separated by 12% denaturing PAGE. The leftmost lane (M) indicates a 23-mer marker. B. caldotenax UvrA and UvrB, and T. maritima UvrC were used in all assays shown in Figure 3. (C) PAGE analysis of the 5′ and 3′ incisions of 60OXA-DPCs. 60OXA-DPCs (5′,3′ end labeled) were incubated with or without UvrABC for 30 min. Products were analyzed as in (B). The two leftmost lanes (M) indicate 23-mer and 25-mer+∗pdC markers. (D) PAGE analysis of the loading of UvrB onto 60OXA-DPCs. 60OXA-DPCs (5′ end labeled) were preincubated with UvrA and/or UvrB. The UvrA-DNA and UvrB-DNA complexes were separated by 4% native PAGE. The concentrations of UvrA were 0, 1, 5, and 10 nM (lanes 1–4). (E) Variation of the incision efficiency of UvrABC with the size of CLPs. The amounts of 5′ incision products were quantified from (B) (incubation time for 30 min) and are plotted against the size of CLPs. (F) Variation of the loading efficiency of UvrB with the size of CLPs. The amounts of the UvrB-DNA complex were quantified from experiments similar to (D) (5 nM UvrA and 100 nM UvrB) and are plotted against the size of CLPs. Molecular Cell 2007 28, 147-158DOI: (10.1016/j.molcel.2007.07.029) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 FA-Induced CLPs Are Removed by NER In Vivo (A) Time courses of the release of CLPs from chromosomal DNA in WT and uvrA cells during postrepair incubation. Cells were exposed to 10 mM FA, and DNA was isolated after the indicated time of repair incubation. Covalently trapped proteins on 15 μg of DNA were labeled by FITC and their fluorescence was measured. Data points are means of three independent experiments with standard deviation. (B) Upper panel, typical SDS-PAGE data of CLPs. Chromosomal DNA (40 μg) isolated as in (A) was heated, and released CLPs were separated by SDS-PAGE. Bands were visualized by silver staining. The two leftmost lanes show size markers. The numbers on the right-hand side of the gel denote the sets of bands analyzed by densitometry. Lower panel, densitometric traces of the gel (upper panel) obtained by NIH Image. Horizontal arrows indicate the sets of bands, and the numbers on the individual peaks show their approximate molecular masses (kDa). (C) Comparison of the percentages of remaining proteins in WT and uvrA cells. The peak areas of band sets in the lower panel of (B) (repair time for 0 and 30 min) were quantified by NIH Image, and the percentages of remaining proteins after 30 min of repair incubation were calculated and are plotted against band sets 1–4. Data points are means of three independent experiments with standard deviation. Molecular Cell 2007 28, 147-158DOI: (10.1016/j.molcel.2007.07.029) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Repair of DPC-Containing Plasmid by NER and the Role of Cytosolic ATP-Dependent Proteases in NER (A) In vivo repair of DPC-containing plasmid. The WT (open bars) and uvrA (closed bars) cells were transformed with pGL3-CMV containing FA-induced DPCs (histone H1 [22 kDa] or digested H1 histones [4.5 kDa and 1.8–3.5 kDa]). The transformation efficiencies (i.e., the repair efficiencies of DPCs) were determined by colony formation and are plotted against the size of CLPs. The leftmost lane (cont.) indicates the results for the unmodified plasmid. Data are means of two independent experiments. (B) Negligible role of cytosolic ATP-dependent proteases in NER. MG1655 (WT), KY2350 (deficient in all cytosolic ATP-dependent proteases), and KY3052 (KY2350 + sulA) were treated with FA (upper panel) or azaC (lower panel), and cell survival was analyzed. Data are means of two independent experiments. (C) Removal of CLPs from chromosomal DNA in FA-treated MG1655, KY2350, and KY3052 cells. CLPs were labeled with FITC and quantified as in Figure 4A. Data points are means of two independent experiments. Molecular Cell 2007 28, 147-158DOI: (10.1016/j.molcel.2007.07.029) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 HR of DPCs Proceeds through the RecBCD Pathway and Requires RuvABC and RecG (A and B) Involvement of the RecBCD, but not the RecFOR, pathway in the HR of DPCs. Cells (WT, recB, and recF) were treated with FA or azaC. (C and D) Indispensable roles of RuvABC and RecG in the HR of DPCs. Cell survival was analyzed as in (A) and (B) with recombination-deficient mutants (ruvA, ruvC, ruvABC, and recG). Data are means of two independent experiments. (E) RecB- and DPC-dependent HR of the plasmid and chromosomal lacZ genes. Cells (WT, recA, recB, and recF) were transformed with unmodified (closed bars) and histone H1-modified (open bars) pCUL-lacZ::kan, and blue and white colonies were counted. The recombination frequencies were calculated as a percentage as 100 × white/(blue + white). Molecular Cell 2007 28, 147-158DOI: (10.1016/j.molcel.2007.07.029) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Possible Pathways of DSB End Formation Associated with DPCs Molecular Cell 2007 28, 147-158DOI: (10.1016/j.molcel.2007.07.029) Copyright © 2007 Elsevier Inc. Terms and Conditions