RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand Crosslink Repair and Human Health

Summary Defects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling. Moreover, single point mutations in the RPA32 subunit of RPA that abolish interaction with RFWD3 also inhibit ICL repair, demonstrating that RPA-mediated RFWD3 recruitment to stalled replication forks is important for ICL repair. We also report that unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells. These data reveal important roles for RFWD3 localization in protecting genome stability and preserving human health.


Supplemental Data
Evidence exists for different modes of ICL repair in S-phase. In one of these models, it has been proposed that ICL repair is initiated by the convergence of two replication forks on the ICL. Various signaling proteins including the Fanconi Anemia (FA) core complex are recruited to the vicinity of the blocked replisome. This triggers the mono-ubiquitylation of FANCD2 and its paralog FANCI at Lys561 and Lys523 respectively, that in turn directs subsequent steps of ICL repair. Cleavage of the leading strand template of one of the forks in concert with cleavage of the same strand on the opposite side of the ICL would unhook the ICL. This results in two one-ended DSBs and a gapped duplex with the ICL adduct on one strand that is filled in by translesion synthesis. The ICL adduct is excised and resection of one of the DSBs generated by unhooking initiates homologous recombination that completes repair. (B) Cells from puromycin-resistant clones obtained after genome editing to disrupt RFWD3 using a gRNA directed to exon 4 were seeded in 96 well plates. Cells were either left untreated, or treated with MMC. After 48 hours, MTS reagent was added and absorbance at 490nm read. Viability of untreated cells was defined as 100%. Only data from cells exposed to MMC is shown.
(C) The sequence of the first mutant allele in RFWD3 Δ/Δ HeLa cells (clone 7) is aligned with the sequence of RFWD3 from control cells. The binding site of the exon 4-targeting gRNA is highlighted in red. The start and end points of  (D) RFWD3 Δ/Δ HeLa cells or parental cells treated were exposed to MMC for the times indicated and extracts were subjected to western blotting with FANCD2 antibodies to monitor FANCD2 ubiquitylation.    Fig. 5 and Fig. 6) (A, B) Control or RFWD3 Δ/Δ HeLa cells were exposed to MMC for 2h, then washed free of drug and allowed to recover for the times indicated. Cells were pre-extracted, fixed and subject to immunofluorescence analysis to measure the number of RPA (A) or RAD51 (B) foci per cell in a field of 100 cells.
Control cells are parental cells that were taken through genome editing protocols but are wild type for RFWD3. Data are represented as mean ± SEM, n=3.
(C) Control or RFWD3 Δ/Δ HeLa cells were exposed to MMC for 2h, then washed free of drug and allowed to recover for the times indicated. Cells were pre-extracted, fixed and incubated with RPA32 and RAD51 primary antibodies conjugated to oligonucleotides. Ligation and amplification with fluorescent dNTPs were then carried out, followed by DAPI staining. Samples were then imaged on a Zeiss 710 confocal. Representative images of the foci observed are shown for each time point.
(D) U2OS cells stably expressing FLAG epitope only (EMPTY), FLAG-RPA32 wild type (WT), or FLAG-RPA32 bearing the mutations indicated were transfected with an siRNA targeting the 3'UTR of the RPA32 gene. "ΔC" refers to an RPA32 deletion mutant truncated from amino acid 242. Cell extracts were subjected to western blotting with the antibodies indicated.