Article Figures & Data
Figures
- FIG. 1.
Examination of extracts from hMsh2-defective cell lines in the CRS assay. (A) The CLT plasmid contains a single psoralen cross-link at the site identified by an “X.” The CT plasmid is identical to the CLT except for the presence of the cross-link. The DT (for donor template) plasmid is identical to the CT plasmid except for the presence of a short segment containing two SspI sites that replace the NcoI site. (B) Silver-stained gels of purified hMutSβ and hMutSα after sodium dodecyl sulfate-PAGE. (C) CRS assay with extracts prepared from HEC59 cells. The assays were performed as described in Materials and Methods. For the complementation assays, purified hMutSβ and hMutSα were added to a final concentration of 0.4 ng/ml. Subsequent to the incubation, the DNA was extracted, digested with NcoI and AvaII, subjected to agarose gel electrophoresis, and analyzed by autoradiography.
- FIG. 2.
Analysis of products created by processing of psoralen ICLs in HeLa extracts. CRS assays were performed as described in Materials and Methods, and extracted DNAs were digested with the indicated restriction enzymes and examined by denaturing PAGE. (A) The local sequence map of the region surrounding the defined ICL in the CLT. Cleavage sites of the appropriate restriction enzymes on both strands of the DNA are indicated by vertical bars. The numbers between the vertical bars indicate the distances in nucleotides between the cleavage sites. The boxed characters represent the thymine nucleotides that are cross-linked by the psoralen adduct. The horizontal bar represents the location of the sequencing primer. (B) Denaturing PAGE analysis of reaction products after digestion by the indicated restriction enzymes. DNAs (CLT and DT) recovered after incubation in HeLa extract were digested with the indicated enzymes (even-numbered lanes). U and L indicate the bands derived from the upper and lower strands, respectively. For size markers, the CT was digested with BsrGI and the appropriate restriction enzyme and radiolabeled by incubation with T4 polynucleotide kinase and [γ-32P]ATP (odd-numbered lanes). Lanes M indicate the 10-bp marker. (C) Determination by sequencing gel analysis of the 3′ terminus of the fragment resulting from incision and DNA synthesis occurring at the site of the psoralen ICL in HeLa extracts. Sequencing reactions (lanes 1 to 4) were performed with the CT plasmid as template and the indicated primer (seeMaterials and Methods). Assays were conducted with the CLT in the absence of the DT. After incubation, the recovered DNA was digested with BssHII, and loaded either alone (lane 6) or in combination with the “G” sequencing reaction (lane 5). Fragments resulting from digestion of the CT with BssHII and BsrGI are also shown as an additional marker (lane 7). The bands derived from the CLT and CT (lanes 6 and 7) are indicated by arrows on the right side of the panel. On the left side of the panel, the cross-linked thymine residue is indicated by an asterisk, and the 3′ end of the reaction product is indicated by an arrow.
- FIG. 3.
DSBs are created at the site of psoralen ICLs in HeLa cell extracts. Arrows to the left in each panel indicate fragments resulting from the formation of DSBs. Restriction enzyme maps are shown to the right of each gel figure, indicating the expected products from a DSB created at the site of the ICL, indicated by an X. Top, CRS assay demonstrating the formation of DSBs in vitro as a function of the concentration of the DT plasmid. After incubation in the extract, recovered DNAs were digested with NcoI and AvaII and examined by agarose gel electrophoresis followed by autoradiography. Bottom, DSB formation at the site of psoralen ICLs in a prelabeled cross-linked substrate requires the HeLa cell extract. CLT or CT plasmids were digested with BssHII and labeled by filling in the 3′ ends with Klenow fragment in the presence of [α-32P]dCTP. The labeled substrates were incubated with HeLa extract in the absence of the DT plasmid. Recovered DNAs were extracted and examined by PAGE followed by autoradiography.
- FIG. 4.
Incisions and DNA synthesis during psoralen ICL processing in vitro are dependent upon Ercc1-Xpf and hMutSβ. Assay were performed as described in the legend to Fig. 2 using the BssHII restriction enzyme, which generates fragments of 113 and 86 nucleotides (see Fig. 2B, lane 2). (A) Assay of extracts from Chinese hamster ovary cells defective in either ERCC1 or XPF. UV20 and UV41 were derived from the repair-normal cell line AA8 used as a control. For complementation assays, purified Ercc1-Xpf was added to a final concentration of 0.5 ng/ml. (B) Assay of extracts from XP lymphoid cell lines defective in XPC (XP1BE) or XPG (XPG83). WI-L2 is a repair-normal human lymphoid cell line used as a control. (C) Assay of extracts from Fanconi anemia lymphoid cell line defective in FANCA (GM13022A), FANCB (GM13071), or FANCC (GM13020). (D) Assay of MMR-deficient human tumor cell lines defective in hMSH2 (HEC59, LoVo), hMSH6 (DLD1), or hMLH1 (SW48, A2780/CP70). For complementation assays, hMutSβ or hMutSα was added to a final concentration of 0.4 ng/ml.
- FIG. 5.
Binding of hMutSβ complex to psoralen DNA ICLs as determined by EMSA. Binding assays were performed as described in Materials and Methods. The positions of the specific (S1 and S2) and nonspecific (NS) complexes are indicated (arrows). (A) hMutSβ binds specifically to DNA containing psoralen ICLs. (B) Effect of ADP and ATP on binding of hMutSβ to psoralen ICLs. (C) PCNA stimulates binding of hMutSβ to psoralen ICLs. A nonspecific band created by incubation of the CL oligo or NCL oligo with PCNA alone is indicated (asterisk). (D) Quantitation of the gel shown in panel C. The S and NS complexes in each lane were quantified by densitometry.
- FIG. 6.
Model for processing of a psoralen DNA ICLs in mammalian cell extracts. An ICL in duplex DNA stimulates cleavage (indicated by arrows) on both sides of the cross-link in one strand (a), creating a gapped intermediate (b). DNA synthesis (dashed line) fills in the gap but is blocked at the adducted thymine in the template strand from further progression (c). A second incision(s) in the opposite strand at either or both of the indicated points (arrows) would result in the formation of DSBs (d).
