Hoogenboom WS et al. (2019), The role of SLX4 and its associated nucleases i...

XB-ART-55566

Nucleic Acids Res 2019 Mar 18;475:2377-2388. doi: 10.1093/nar/gky1276.

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The role of SLX4 and its associated nucleases in DNA interstrand crosslink repair.

Hoogenboom WS , Boonen RACM , Knipscheer P .

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A key step in the Fanconi anemia pathway of DNA interstrand crosslink (ICL) repair is the ICL unhooking by dual endonucleolytic incisions. SLX4/FANCP is a large scaffold protein that plays a central role in ICL unhooking. It contains multiple domains that interact with many proteins including three different endonucleases and also acts in several other DNA repair pathways. While it is known that its interaction with the endonuclease XPF-ERCC1 is required for its function in ICL repair, which other domains act in this process is unclear. Here, we used Xenopus egg extracts to determine ICL repair specific features of SLX4. We show that the SLX4-interacting endonuclease SLX1 is not required for ICL repair and demonstrate that all essential SLX4 domains are located at the N-terminal half of the protein. The MLR domain is crucial for the recruitment of XPF-ERCC1 but also has an unanticipated function in recruiting SLX4 to the site of damage. Although we find the BTB is not essential for ICL repair in our system, dimerization of SLX4 could be important. Our data provide new insights into the mechanism by which SLX4 acts in ICL repair.

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Species referenced: Xenopus laevis
Genes referenced: ercc1 ercc4 slx4 terf2

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	Figure 1. SLX4 depletion inhibits ICL repair in Xenopus egg extracts. (A) Schematic illustration of the domain organization and interaction partners of SLX4 mapped on the Xenopus laevis SLX4 sequence. Protein interactions are represented by dashed lines. Interaction partners with unknown SLX4 interaction sites are shown in grey. Endonucleases are marked with scissors. Domain boundaries are based on previous reports (28,29,31,33,62). Interaction sites that do not align well with the Xenopus laevis sequence are not numbered and may not be conserved. Especially TRF2 interaction may be human specific (56). (B) Mock- and SLX4-depleted nucleoplasmic egg extract (NPE) were analyzed by western blot using Î±-SLX4 antibody. A dilution series of undepleted extract was loaded on the same blot to determine the degree of depletion. A relative volume of 100 corresponds to 0.4 Î¼l NPE. (C) As in (B) but using Î±-XPF (upper panel) or Î±-ERCC1 antibody (lower panel). (D) Mock-depleted (Mock), SLX4-depleted (ÎSLX4), and SLX4-depleted NPE complemented with wild-type SLX4 (ÎSLX4+SLX4WT) were analyzed by western blot using Î±-SLX4 antibody (right panel). These extracts, with SLX4-depleted high-speed supernatant (HSS) extract, were used to replicate pICL. Repair efficiency was calculated and plotted (left panel). A higher than endogenous concentration of recombinant SLX4 was required for complete rescue, likely due to partially loss of function of the recombinant protein during purification. (E) SLX4-depleted (ÎSLX4), and SLX4-depleted NPE complemented with wild-type SLX4 (ÎSLX4+SLX4WT) or wild-type SLX4 and XPF-ERCC1 (ÎSLX4+SXE) were analyzed by western blot using Î±-SLX4 or Î±-XPF antibodies (right panel). These extracts, with SLX4-depleted HSS, were used to replicate pICL. Repair efficiency was calculated and plotted (left panel). Line within blot indicates position where irrelevant lanes were removed. *, background band. #, SapI fragments from contaminating uncrosslinked plasmid present in varying degrees in different pICL preparations. See also Supplementary Figure S1.
	Figure 2. SLX1 is not required for ICL repair in Xenopus egg extracts. (A) Schematic illustration of the xlSLX4SBD* mutant protein. The C1753R mutation, indicated by a red line, is predicted to disrupt interaction with SLX1. (B) SLX4-depleted (ÎSLX4), and SLX4-depleted NPE complemented with wild-type SLX4 (ÎSLX4+SLX4WT) or mutant SLX4 (ÎSLX4+SLX4SBD) were analyzed by western blot using Î±-SLX4 antibody (right panel). These extracts, with SLX4-depleted HSS, were used to replicate pICL. Repair efficiency was calculated and plotted (left panel). Line within blot indicates position where irrelevant lanes were removed. (C) SLX1, wild-type and mutant SLX4 (SLX4WT and SLX4SBD respectively) were individually expressed in Sf9 insect cells. Cells were lysed and indicated lysates were mixed and incubated. SLX1 was immunoprecipitated using PAS-conjugated Î±-SLX1 antibodies. The input (IN), flow-through (FT), and bound fractions (B) were analyzed by western blot using Î±-FLAG (upper panel) and Î±-SLX1 antibodies (lower panel). Line within blot indicates position where irrelevant lanes were removed. (D) Mock- and SLX1-depleted NPE were analyzed by western blot using Î±-SLX1 (upper panel) and Î±-SLX4 antibodies (lower panel). A dilution series of undepleted extract was loaded on the same blots to determine the degree of depletion. A relative volume of 100 corresponds to 0.4 Î¼l NPE. (E) Mock- and SLX4-depleted NPE were analyzed by western blot using Î±-SLX1 antibody. A dilution series of undepleted extract was loaded on the same blot to determine the degree of depletion. A relative volume of 100 corresponds to 0.4 Î¼l NPE. (F) Mock-depleted (Mock), SLX1-depleted (ÎSLX1), and SLX1-depleted NPE complemented with wild-type SLX4 (ÎSLX1+SLX4WT) or SLX4-SLX1 complex (ÎSLX4+SLX4/1) were analyzed by western blot using Î±-SLX1 (upper right panel) and Î±-SLX4 antibodies (lower right panel). These extracts, with SLX1-depleted HSS, were used to replicate pICL. Repair efficiency was calculated and plotted (left panel). *, background band. #, SapI fragments from contaminating uncrosslinked plasmid present in varying degrees in different pICL preparations. See also Supplementary Figure S2.
	Figure 3. The N-terminal half of SLX4 is sufficient to support ICL repair. (A) Schematic illustration of the xlSLX41â840 mutant protein. This protein is truncated at residue 840. (B) SLX1, wild-type SLX4 (SLX4WT) and SLX4 mutants (SLX4MUT) were individually expressed in Sf9 insect cells. Cells were lysed and indicated lysates were mixed and incubated. SLX4 was immunoprecipitated using PAS-conjugated Î±-SLX4 antibodies. The input (IN), flow-through (FT), and bound fractions (B) were analyzed by western blot using Î±-FLAG (upper panel) and Î±-SLX1 antibodies (lower panel). Line within blot indicates position where irrelevant lanes were removed. (C) SLX4-depleted (ÎSLX4), and SLX4-depleted NPE complemented with wild-type SLX4 (ÎSLX4+SLX4WT) or mutant SLX4 (ÎSLX4+SLX41â840), were used with SLX4-depleted HSS to replicate pICL. Repair efficiency was calculated and plotted (left panel). Protein dilutions of wild-type SLX4 (SLX4WT) and mutant SLX4 (SLX41â840) were analyzed by western blot using Î±-FLAG antibody to ensure equivalent protein concentrations (right panel). *, background band. #, SapI fragments from contaminating uncrosslinked plasmid present in varying degrees in different pICL preparations. See also Supplementary Figure S3.
	Figure 4. The MLR domain ensures XPF-binding and contributes to SLX4-recruitment to ICLs. (A) Schematic illustration of the xlSLX4ÎMLR mutant protein. The residues 286â444, including the MLR domain, are replaced by a short linker, indicated by a black line. This deletion is predicted to disrupt interaction with XPF-ERCC1. (B) Schematic illustration of the xlSLX4MLR* mutant protein. The four point mutations in the MLR domain, T418A, W420A, Y434A, and Y435A, are indicated by two red lines and predicted to disrupt interaction with XPF-ERCC1. (C) SLX4-depleted (ÎSLX4), and SLX4-depleted NPE complemented with wild-type SLX4 (ÎSLX4+SLX4WT) or mutant SLX4 (ÎSLX4+SLX4ÎMLR) were analyzed by western blot using Î±-SLX4 antibody (right panel). These extracts, with SLX4-depleted HSS, were used to replicate pICL. Repair efficiency was calculated and plotted (left panel). Line within blot indicates position where irrelevant lanes were removed. (D) SLX4-depleted (ÎSLX4), and SLX4-depleted NPE complemented with wild-type SLX4 (ÎSLX4+SLX4WT) or mutant SLX4 (ÎSLX4+SLX4ÎMLR) were analyzed by western blot using Î±-SLX4 (upper right panel) and Î±-XPF antibodies (lower right panel). These extracts, with SLX4-depleted HSS, were used to replicate pICL. Samples were taken at various times and immunoprecipitated with Î±-XPF (left panel) or Î±-SLX4 antibodies (middle panel). Co-precipitated DNA was isolated and analyzed by quantitative PCR using the pICL or pQuant primers. Recovery values of pQuant were subtracted from pICL recovery values. The resulting data were plotted as the percentage of peak value with the highest value within one experiment set to 100%. (E) Purified wild-type SLX4 (SLX4WT) or mutant SLX4 (SLX4MUT) were added to HSS. After incubation, recombinant SLX4 was immunoprecipitated using anti-FLAG M2 affinity gel (Sigma). The input (IN), flow-through (FT), and bound fractions (B) were analyzed by western blot using Î±-FLAG (upper panel) and Î±-XPF antibodies (lower panel). Line within blot indicates position where irrelevant lanes were removed. (F) As in (D) but using the xlSLX4MLR* mutant protein. (G) As in (C) but using the xlSLX4MLR* mutant protein. Line within blot indicates position where irrelevant lanes were removed. *, background band. #, SapI fragments from contaminating uncrosslinked plasmid present in varying degrees in different pICL preparations. See also Supplementary Figure S4.
	Figure 5. The BTB domain is not absolutely required for ICL repair but contributes to SLX4 dimerization. (A) Schematic illustration of the xlSLX4BTB* mutant protein. The two point mutations in the BTB domain, F644A and Y646A, are indicated by a red line and predicted to affect SLX4 dimerization and XPF positioning. (B) Schematic illustration of the xlSLX41â558 mutant protein. This protein is truncated at residue 558. (C) Purified wild-type SLX4 (SLX4WT), mutant SLX4 (SLX4BTB) or buffer (-) were added to HSS. After incubation, recombinant SLX4 was immunoprecipitated using anti-FLAG M2 affinity gel (Sigma). The input (IN), flow-through (FT), and bound fractions (B) were analyzed by western blot using Î±-FLAG (upper panel) and Î±-XPF antibodies (lower panel). Line within blot indicates position where irrelevant lanes were removed. (D) SLX4-depleted (ÎSLX4), and SLX4-depleted NPE complemented with wild-type SLX4 (ÎSLX4+SLX4WT) or mutant SLX4 (ÎSLX4+SLX4BTB) were analyzed by western blot using Î±-SLX4 antibody (right panel). These extracts, with SLX4-depleted HSS, were used to replicate pICL. Repair efficiency was calculated and plotted (left panel). Line within blot indicates position were irrelevant lanes were removed. (E) Wild-type (SLX4WT) or mutant SLX4 (SLX4BTB) containing an N-terminal FLAG-tag and a C-terminal Strep-tag, were co-expressed with wild-type SLX4 containing an N-terminal His-tag and a C-terminal Strep-tag (His-SLX4) in Sf9 insect cells. Cells were lysed and FLAG-tagged SLX4 was immunoprecipitated using anti-FLAG M2 affinity gel (Sigma). To examine the contribution of DNA to SLX4-dimerization the cell lysates were split during the immunoprecipitation step and treated with benzonase (Sigma) (+) or buffer (â). The input (IN), flow-through (FT), and bound fractions (B) were analyzed by western blot using Î±-FLAG and Î±-His antibodies. Line within blot indicates position where irrelevant lanes were removed. (F) SLX4-depleted (ÎSLX4), and SLX4-depleted NPE complemented with wild-type SLX4 (ÎSLX4+SLX4WT) or mutant SLX4 (ÎSLX4+SLX41â558), were used with SLX4-depleted HSS to replicate pICL. Repair efficiency was calculated and plotted (left panel). Protein dilutions of wild-type SLX4 (SLX4WT) and mutant SLX4 (SLX41â558) were analyzed by western blot using Î±-FLAG antibody to ensure equivalent protein concentrations (right panel). (G) As in (E) but using the xlSLX41â558 mutant protein and without benzonase treatment. , background band. #, SapI fragments from contaminating uncrosslinked plasmid present in varying degrees in different pICL preparations. See also Supplementary Figure S5.

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