Figure 7. SLX1-SLX4 complexes resolve HJs through an ordered pathway in vitro and are required for efficient homology-directed repair of DSBs in vivo.
(A) The indicated SLX4 and GEN1 complexes were incubated with an X26 substrate +/−T4 DNA ligase prior to separation on a denaturing gel and autoradiography.
(B) The indicated SLX4-HA immune complexes were immunoblotted for ERCC4 and MUS81.
(C) Recombinant SLX1-SLX4SBD complexes generate cleaved X26 structures that are ligatable.
(D) Requirement of SLX1 and MUS81 for cleavage of static HJs. 293T/SLX4-HA cells were transfected with control siRNA or siRNAs targeting SLX1 (SLX1#4) or MUS81 (MUS81-17). After 72 h, SLX4-HA complexes were assayed in cleavage assays using HJ X0-BE substrates labeled on either strand 1 or strand 3. Products were resolved on denaturing gels prior to autoradiography.
(E) SLX4-HA immune complexes corresponding to panel D were immunoblotted for ERCC4 and MUS81.
(F) qPCR analysis of mRNA isolated from cells transfected with control siRNA or siRNA targeting SLX1 (#4). mRNA abundance is relative to GAPDH.
(G–I) MUS81-EME1 complexes cleave nicked HJs that mimic those produced by SLX1-SLX4. Bacterial GST-MUS81-His6-EME1 complexes were incubated (37°C, 30 min) with synthetic nicked HJs (XO1 strand nicked at nucleotide 32) labeled on either strand 1 or strand 3 and the products analyzed on either native (panel G) or denaturing (panel H) gels. The positions of cleavage of the nicked XO structure labeled on strand 3 are shown in panel I.
(J) GST-MUS81-His6-EME1 (12.5, 25, 50 ng) was incubated (37°C, 30 min) with XO-BE in the presence or absence of 3 ng of recombinant SLX1-SLX4SBD, and the products analyzed on denaturing gels.
(K) DR-GFP U2OS were infected with viral vectors expressing the indicated shRNAs followed by I-SceI expression 48h later. The extent of homologous recombination was determined by flow cytometry. *, p < 0.05; students T-test. Error bars are STDEV across 3 technical replicates.
(L) Model depicting distinct roles of SLX1 and MUS81 within the SLX4 complex for HJ cleavage (see text for details).