Targeting the FANCJ-BRCA1 interaction promotes a switch from recombination to poleta-dependent bypass

Abstract

BRCA1 and the DNA helicase FANCJ (also known as BACH1 or BRIP1) have common functions in breast cancer suppression and DNA repair. However, the functional significance of the direct interaction between BRCA1 and FANCJ remains unclear. Here, we have discovered that BRCA1 binding to FANCJ regulates DNA damage repair choice. Thus, when FANCJ binding to BRCA1 is ablated, the molecular mechanism chosen for the repair of damaged DNA is dramatically altered. Specifically, a FANCJ protein that cannot be phosphorylated at serine 990 or bind BRCA1 inhibits DNA repair via homologous recombination and promotes poleta-dependent bypass. Furthermore, the poleta-dependent bypass promoted by FANCJ requires the direct binding to the mismatch repair (MMR) protein, MLH1. Together, our findings implicate that in human cells BRCA1 binding to FANCJ is critical to regulate DNA repair choice and promote genomic stability. Moreover, unregulated FANCJ function could be associated with cancer and/or chemoresistance.

Figure 1

FANCJ^S990A, as compared with FANCJ^WT, promotes a distinct DNA damage response in FA-J cells. (a) FANCJ-null FA-J cells were complemented with vector, FANCJ^WT, or FANCJ^S990A and lysates were analyzed by immunoblot with the indicated antibodies (Abs). (b) The FA-J cell lines were treated constitutively with 0.25 µg/ml melphalan, collected at the indicated times, and analyzed by FACS to determine the percentage of cells in G2/M. A representative experiment is shown. The bar graph represents mean ± s.d. from three independent experiments. The asterisk indicates a significant difference (P<0.05, unpaired t-test). (c) The FA-J cells expressing vector, FANCJ^WT, or FANCJ^S990A were plated at low density, treated with the indicated doses of cisplatin, UV, zeocin, 6-thioguanine, or methyl methanesulfonate and allowed to grow for 5–8 days. The cells were then collected and counted to analyze percent growth. Data represent mean percent ± s.d. of growth from three independent experiments.

Figure 2

FANCJ^S990A reduces DSB-induced RAD51 foci and HR. (a) The FA-J cell lines were treated with 12.5 µg/ml of zeocin and immunofluorescence was performed with γ-H2AX and RAD51 Abs. A representative image is shown to depict how staining was observed. (b) The γ-H2AX and (c) RAD51 foci were quantitated based on a cell being positive (> 10) foci per 300 DAPI positive cells from three independent experiments. Asterisk indicates significant difference (P<0.05, unpaired t-test). (d) DR-U2OS cells were cotransfected with the I-Sce-1 endonuclease and vector, FANCJ^WT, FANCJ^S990A, or FANCJ^K52R, collected and either lysed and immunoprecipitated followed by immunoblot with the indicated Abs or analyzed by FACS. The bar graph shows the percentage of GFP-positive cells.

Figure 3

FANCJ enhances DNA damage-induced polη foci. (a) U2OS cells were cotransfected with siRNA for luc or Rad18 and eGFP-polη and either collected for immunoblot with the indicated Abs or UV irradiated and assessed for eGFP-polη foci. Cells were assessed for eGFP-polη foci by autofluorescence. (b) U2OS cells stably expressing vector, FANCJ^S990A, or FANCJ^WT were transfected with eGFP-polη and either collected for immunoblot with the indicated Abs or UV irradiated with indicated dose with 4 h incubation or treated with 250 nm MMC with incubation at varying times. Data represent the mean percent ± s.d. cells positive (> 10) green foci per 300 DAPI positive cells from three independent experiments. Asterisk indicates significant difference (P<0.05, unpaired t-test).

Figure 4

FANCJ^S990A promotes ICL and UV resistance in a polη-dependent manner. (a) FA-J cells stably expressing vector, FANCJ^WT, or FANCJ^S990A (Figure 1a) were transfected with siRNA to Luc or polη, incubated for 48 h, treated with MMC or UV, and percent growth was assessed as in Figure 1. (b) FA-J cell lines transfected with siRNA to luc, polη, polη #1, or polη #2 were collected for immunoblot with the indicated Abs. (c) The FA-J cell lines with indicated siRNAs were incubated for 48 h, treated with MMC at the IC50 dose, and percent growth was assessed as in Figure 1. Graph shows the percent growth mean±s.d. (d) FA-J cells stably expressing vector, FANCJ^WT, or FANCJ^S990A were transfected with siRNA to luc, Rad18, Rad54, or Rev1 and incubated for 48 h, treated with MMC, and percent growth was assessed as in Figure 1. (e) Cells used in (d) were collected and lysed for immunoblot with the indicated Abs.

Figure 5

FANCJ^S990A requires MLH1 binding to promote polη-dependent bypass. (a) FA-J cells stably expressing vector, FANCJ^WT, FANCJ^S990A, or FANCJ^K141/142A were collected and immunoprecipitated with anti-FANCJ Abs and blotted with the indicated Abs. (b) FA-J cells stably expressing vector, FANCJ^WT, FANCJ^S990A, FANCJ^K141/142A, or FANCJ^{K141/142A/S990A} were either collected for immunoblot with the indicated Abs or (c) treated with the indicated doses of MMC and allowed to grow for 5–8 days. The cells were then collected and counted to analyze percent growth. Data represent mean percent ± s.d. of growth from three independent experiments. (d) The FA-J cell lines were treated constitutively with 0.25 µg/ml melphalan, collected at the indicated times, and analyzed by FACS to determine the percentage of cells in G2/M. A representative experiment is shown. The bar graph represents mean ± s.d. from three independent experiments. (e) Model summarizes observations of this study. FANCJ when uncoupled from BRCA1 promotes polη-dependent TLS in a manner that requires MLH1 binding. Dotted line to MMR is added as a discussion point. To promote TLS, FANCJ could limit negative regulators of TLS, such as MMR.