Opposing Roles of FANCJ and HLTF Protect Forks and Restrain Replication during Stress

Abstract

The DNA helicase FANCJ is mutated in hereditary breast and ovarian cancer and Fanconi anemia (FA). Nevertheless, how loss of FANCJ translates to disease pathogenesis remains unclear. We addressed this question by analyzing proteins associated with replication forks in cells with or without FANCJ. We demonstrate that FANCJ-knockout (FANCJ-KO) cells have alterations in the replisome that are consistent with enhanced replication stress, including an aberrant accumulation of the fork remodeling factor helicase-like transcription factor (HLTF). Correspondingly, HLTF contributes to fork degradation in FANCJ-KO cells. Unexpectedly, the unrestrained DNA synthesis that characterizes HLTF-deficient cells is FANCJ dependent and correlates with S1 nuclease sensitivity and fork degradation. These results suggest that FANCJ and HLTF promote replication fork integrity, in part by counteracting each other to keep fork remodeling and elongation in check. Indicating one protein compensates for loss of the other, loss of both HLTF and FANCJ causes a more severe replication stress response.

Keywords: DNA replication; FANCJ/BACH1/BRIP1; Fanconi anemia; fork degradation; fork protection; helicase; hereditary breast cancer; iPOND; replication stress response; replisome.

Figure 1.. FANCJ and Its Helicase Activity Protect Nascent DNA at Replication Forks from MRE11-Dependent Degradation

(A) Western blot analysis with the indicated antibodies (Abs) of lysates from control and FANCJKO 293T cells. (B) Schematic, representative images, and quantification of the CldU/IdU ratio after HU treatment with or without mirin. (C) Western blot analysis with indicated Abs of lysates from FANCJ-null FA-J cells complemented with vector (V), wild-type (WT), or a catalytically inactive FANCJ (K52R) mutant. (D) Schematic, representative images, and quantification of the CldU/IdU ratio after HU treatment. Each dot represents one fiber. For each analysis, at least 200 fibers are quantified from two independent experiments. Red bars represent the median. Statistical analysis according to two-tailed Mann-Whitney test; ****p < 0.0001. Scale bars, 10 μm.

Figure 2.. Isolation of Proteins on Nascent DNA (iPOND) from FANCJ-KO and Control 293T Cells

(A) Western blot analysis with the indicated Abs of input or iPOND samples following non-click, click, or thymidine chase from 293T cells. (B) Western blot analysis with indicated Abs of input or iPOND samples from control and FANCJ-KO 293T cells. (C) Schematic representation of the SILAC iPOND technique. (D) Volcano plots of the p values versus the log2 protein abundance differences between purified replisomes of 293T control (heavy) and FANCJ-KO (light) cells. Significantly enriched and reduced proteins are highlighted in red and blue, respectively. p values are calculated from the data of three biological repeats. (E) Table shows the light/heavy ratio in chromatin fractions for indicated proteins. (F) Western blot analysis with the indicated Abs of WCE and chromatin preparations from 293T cells. (G) Western blot analysis with the indicated Abs of cells transfected with vector (V) or wild-type FANCJ (WT) 293T cells. Asterisk indicates nonspecific band.

Figure 3.. HLTF Contributes to Fork Degradation in FANCJ-KO Cells, and FANCJ Contributes to Fork Degradation in HLTF-Depleted Cells following Prolonged Stress

(A) Western blot analysis with the indicated Abs of lysates from control and FANCJ-KO 293T cells expressing shRNA against HLTF or NSC. (B) Schematic, representative images, and quantification of the CldU/IdU ratio after HU treatment. (C) Schematic, representative images, and quantification of the CIdU tract length. Each dot represents one fiber; at least 200 fibers are quantified from two independent experiments. Red bars represent the median. Statistical analysis according to two-tailed Mann-Whitney test; ****p < 0.0001.

Figure 4.. HLTF Counteracts Replication Stress in FANCJ-KO Cells

(A) Western blot analysis with the indicated Abs of lysates from control and FANCJ-KO U2OS cells and when expressing shRNA against HLTF or NSC. (B) Cell survival assays with FANCJ-KO and control U2OS cells expressing shRNA against HLTF or NSC under increasing concentrations of MMC or HU. Data represent the mean percent ± SD of survival from three independent experiments. (C) γ-H2AX/EdU ratio in FANCJ-KO and control U2OS cells expressing shRNA against HLTF or NSC following treatment with HU. At least 200 cells were measured for each. The assay was completed in triplicate for each shRNA. (D) Quantification of the percentage of comet tail DNA in FANCJ-KO and control 293T cells expressing shRNA against HLTF or NSC following treatment with HU, and images of comet tail formation. 100 cells were measured for each. Statistical analysis according to two-tailed Mann-Whitney test; ****p < 0.0001; *p < 0.05.

Figure 5.. FANCJ Contributes to Unrestrained Replication and S1 Nuclease Sensitivity in HLTF-Depleted Cells

(A) Western blot analysis with the indicated Abs of lysates from HCT116 cells expressing shRNA against FANCJ, HLTF, or NSC. (B) Schematic, representative images, and quantification of CldU tract length during HU treatment. (C) Schematic and quantification of the CldU/IdU ratio in the indicated shRNA-expressing HCT116 cells with or without S1 nuclease incubation. Each dot represents one fiber; at least 200 fibers are quantified from two independent experiments. Red bars represent the median. Statistical analysis according to two-tailed Mann-Whitney test; ****p < 0.0001. (D) Model of opposing roles of FANCJ and HLTF at forks.