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DNA interstrand crosslinks (ICLs) are cytotoxic lesions that covalently link opposite strands of the DNA helix and block DNA unwinding. ICLs are repaired during and outside S phase, and replication-independent ICL repair (RIR) is critical to maintain genomic integrity and to allow transcription in nondividing or slowly dividing cells. Here, we show that the Y family DNA polymerase kappa (Pol κ) is essential for RIR of a site-specific ICL lesion in Xenopus egg extracts, and that both its catalytic activity and UBZ domains are required for this function. We also demonstrate a requirement for PCNA and its modification on lysine 164. Finally, we show that Pol κ participates in ICL repair in mammalian cells, particularly in G0. Our results identify key components of the RIR pathway and begin to unravel its mechanism.
Figure 1. Replication-Independent ICL Repair Occurs via a Mechanism that Is Distinct from Replication-Dependent ICL Repair
(A) Schematic representation of the ICL plasmid. Regions amplified by the X and C primers are illustrated.
(B) ICL plasmid was incubated in HSS (nonreplicating) or HSS + NPE (replication-competent) extract for the times indicated. Repair (%) is measured as the fraction of the X:C ratio of the ICL plasmid over the X:C ratio of the control plasmid. See also Figure S1.
(C) Quantification of ICL repair in mock- and FANCI-depleted HSS or in HSS treated with 100 μM curcumin.
(D) Quantification of ICL repair in mock- and Rad51-depleted HSS.
(E) Quantification of ICL repair in mock- and Rev7-depleted HSS. Results represent the mean and SEM from at least three independent experiments.
Figure 2. Pol κ Is Required for RIR
(A) ICL repair in mock-, Pol κ-, and Pol κ-depleted HSS supplemented with recombinant Pol κWT or Pol κD199A, E200A. See also Figure S2.
(B) Schematic showing the domain structure of Pol κ and the location of mutations used in this study. Asterisks mark the location of the mutations shown in bold and underlined in the table below.
(C) Quantification of ICL repair in mock-depleted HSS, Pol κ-depleted HSS, and Pol κ-depleted HSS supplemented with recombinant Pol κF562A, F563A.
(D) ICL and control plasmids were incubated in Pol κ-depleted HSS, supplemented with either Pol κWT or Pol κD199A, E200A and anaylzed by Pol κ ChIP. The data are expressed as the fold enrichment of DNA pulled down from ICL plasmid compared to control plasmid. A value of 1 (dashed line) indicates no enrichment. Results represent the mean and SEM from at least three independent experiments.
Figure 3. PCNA Modification on Lysine 164 and Pol κ UBZ Domains Are Required for RIR
(A) Quantification of ICL repair in mock- and Rad1-depleted HSS. See also Figure S3.
(B) Quantification of ICL repair in mock-depleted HSS, PCNA-depleted HSS, and PCNA-depleted HSS supplemented with PCNAWT or PCNAK164R.
(C) Control or ICL plasmids were incubated in extract for 40 min and immobilized using biotinylated LacR protein. DNA-bound proteins were analyzed by western blotting.
(D) Quantification of ICL repair in mock-, Pol κ-, or Pol κ-depleted HSS supplemented with Pol κWT, Pol κD634A,D789A, or Pol κI857A,F860A,F861A, as indicated. Results represent the mean and SEM from at least three independent experiments.
Figure 4. Pol κ−/− MEF Sensitivity to Crosslinking Agents Correlates with a Defect in ICL Repair
(A) Schematic of GFP-based ICL assay.
(B) Quantification of the repair of a single site-specific ICL in either Pol κ−/− or Pol κ+/+ MEFs using the GFP-based assay shown in (A). Repair is expressed as the fraction of GFP-positive cells transfected with the ICL plasmid over GFP-positive cells transfected with the control plasmid.
(C) Clonogenic survival assays with Pol κ−/− or Pol κ+/+ MEFs in the presence of the indicated concentration of MMC. Cells were treated during exponential growth or during G0 arrest.
(D) Clonogenic survival assay with Pol κ−/− or Pol κ+/+ MEFs in the presence of the indicated concentration of cisplatin during exponential growth. Results represent the mean and SEM from at least three independent experiments.
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