Räschle M , Knipscheer P , Knipsheer P , Enoiu M , Angelov T , Sun J , Griffith JD , Ellenberger TE , Schärer OD , Walter JC .

GM31819 NIGMS NIH HHS , GM55390 NIGMS NIH HHS , GM62267 NIGMS NIH HHS , R01 GM062267-06 NIGMS NIH HHS , R01 GM062267-07 NIGMS NIH HHS , R01 GM062267-08 NIGMS NIH HHS , R01 GM062267 NIGMS NIH HHS , R01 GM031819 NIGMS NIH HHS , R01 GM055390 NIGMS NIH HHS

	Figure 1 DNA Replication Forks Converge on an Interstrand Crosslink (A) Structure of a nitrogen mustard-like ICL (postulated). (B) Structure of a cisplatin ICL (based on Huang et al., 1995). (C) Replication of pICLNm in Xenopus egg extracts. pCtr or pICLNm was incubated sequentially with HSS and NPE/32P-α-dATP. At the indicated times after NPE addition, replication products were analyzed on a native agarose gel. (D) The average replication efficiency of three independent experiments was plotted with error bars. (E) Model for replication of pICL. (F) pICLNm was replicated as in (C). Thirty minutes after NPE addition, DNA was analyzed by electron microscopy. The predominant species, a “Figure 8” structure, is shown.
	Figure 2 Multistep Lesion Bypass of an Interstrand Crosslink (A) Structure of the replicated AflIII fragment that includes the ICL. S, primer used to generate the sequencing ladder shown in (B). (B) Mapping of nascent strands during replication of pICLNm or pCtr (final concentration 1.2 ng/μl). At the indicated times after NPE addition, reaction products were digested with AflIII and analyzed on a sequencing gel alongside a sequencing ladder derived from extension of primer S on pCtr (see A). Numbers to the left indicate the sizes of the sequencing products. Leading and lagging strands for the rightward and leftward forks are indicated. Square brackets show the positions of leading strands after the initial pausing, whereas red and green arrowheads show their location after advancing toward the ICL. Open arrowhead, extension product. (C) Enlarged and darker exposure of the bottom part of the autoradiogram shown in (B). The most prominent species observed at 45 min are indicated on the right, with the predicted last nucleotide denoted by a single letter. The exact sizes of species T147-A150 were confirmed in Figure S9. (D) Cartoon-form depiction of the results in (C).
	Figure 3 Detection of Incisions near the ICL (A) Expected intermediates resulting from single or dual incisions near the ICL (see text). Note that the first incision could also occur to the left of the ICL, giving rise to a short arm and large Y structure. (B) pICLNm replication products were digested with HincII and separated on a native agarose gel (lanes 1–8). Replicated pCtr was digested with HincII to generate a 5.6 kb size marker (lane 9, only 20% of the reaction loaded). (C) Advance of the leading strand to the ICL precedes incisions. At each time point in (B), the relative abundance of X-shaped molecules was quantified using a phosphorimager (blue line). At 18 and 22 min, before replication was complete, the level was assigned a value of 100. The relative abundance of leading strand products from both forks at the −1 and −4 positions (Figure 2B) (gray dashed line) is plotted. The graph shows the average of four independent experiments with error bars.
	Figure 4 Replication-Dependent Repair of an ICL (A) An ICL blocks cleavage by AccI. Fifteen nanograms of pICLNm or pCtr was digested with AccI, separated on a native agarose gel, and stained with SYBR Gold. (B) ICL repair assay. At different times after addition of NPE/32P-α-dATP, pICLNm or pCtr was recovered and equal aliquots were digested with HincII (lanes 1–9) or AccI (lanes 10–18). (Note: lanes 1–9 represent a darker exposure of Figure 3B.) Twenty percent of the reaction was loaded in lanes 9 and 18. (C) At each time point shown in (B), the repair efficiency was calculated as explained in the text and graphed (red line). Extension products from the same experiment shown in Figure 4B were plotted for comparison (gray dashed line). (D) pICLNm or pCtr was replicated using NPE lacking radioactivity and optionally supplemented with p27Kip. Plasmid was recovered, digested as indicated, and examined by Southern blotting using pCtr DNA as probe. Twenty percent of the reaction was loaded in lane 13. Samples were supplemented with a 1.2 kb HindIII fragment of pCtr before extraction (loading control).
	Figure 5 An Adduct Persists in the Parental Strand after Lesion Bypass (A) Cartoon illustrating replication of an AflIII/AseI restriction fragment harboring a cisplatin ICL. Due to the different overhangs generated by these enzymes, digestion of pICLPt yields top and bottom strands of 178 and 176 nt, respectively. Lesion bypass by the rightward fork yields a radioactively labeled nascent top strand and an adducted, parental bottom strand (Bottom-AD), while lesion bypass by the leftward fork results in a labeled nascent bottom strand and an adducted parental top strand (Top-AD). Strand-specific Southern blotting was used to detect either the top strands (blue lines) or the bottom strands (green lines). (B) Detection of nascent strands. pICLPt or pCtr was replicated in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of 32P-α-dATP. After 4 hr, replication products were digested with AflIII and AseI, separated on a 5% denaturing polyacrylamide gel and transferred to a Nylon membrane. Radioactive products were visualized using a phophorimager. (C) Detection of the nascent top strand and the adducted parental top strand (Top-AD) on the membrane in (B) by Southern blotting using a bottom-strand probe. (D) Detection of the nascent bottom strand and the adducted parental bottom strand by stripping and reprobing the membrane in (C) using a top-strand probe. Primer S was used to generate a sequencing ladder from pCtr that serves as a size marker (see Figure 2). Green and blue arrowheads indicate the 176 nt and 178 nt sequencing products, respectively (see Figure S5C for sequence information and location of primer S). The migration of the digested DNA replication products is retarded by 1 nt with respect to the sequencing products (See Figure S9 for discussion of this effect). Adducted parental strands also persisted on pICLNm (data not shown).
	Figure 6 Repair of pICL Is Defective in Rev7-Depleted Extracts (A) Rev7 immunodepletion. Undepleted, mock-depleted, and Rev7-depleted HSS and NPE were analyzed by western blotting using Rev7 antibody. A relative volume of 100 corresponds to 0.3 μl extract. (B) Accumulation of a new lesion bypass intermediate in Rev7-depleted extracts. pICLPt was replicated in mock-depleted or Rev7-depleted HSS and NPE (4 ng/μl final DNA concentration). At the indicated times, products were digested with AflIII and analyzed on a sequencing gel (as in Figure S7B). Numbers to the left indicate the size of the sequencing products. The new replication intermediate is indicated (0 product). (C) The average repair efficiency in mock- and Rev7-depleted extracts in four independent experiments is plotted with error bars. (D) Cartoon depicting the intermediate that accumulates in Rev7-depleted extracts. We infer that a C residue is inserted at position 145 since the translesion step is likely performed by the cytidyl transferase Rev1, and some of the products are digestible with SapI.
	Figure 7 Model for ICL Repair in Xenopus Egg Extracts When DNA containing an ICL (A) undergoes DNA replication, the leading strands of two converging replication forks initially stall 20–40 nt from the lesion (B). One leading strand (indicated in red) is then extended to within 1 nt of the ICL, a step which may require prior replisome remodeling (C). Subsequently, the two sister chromatids are uncoupled via dual incisions (yellow scissors) on either side of the ICL, possibly by XPF and/or Mus81 (D). Next, a translesion DNA polymerase (possibly Rev1) inserts a nucleotide across from the adducted base (E), after which DNA polymerase ζ extends the nascent strand beyond the ICL (F). Finally, two fully repaired DNA duplexes are generated through the action of nucleotide excision repair (NER) on the top duplex and homologous recombination (HR) on the bottom duplex (G).

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