Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities - PubMed (original) (raw)

Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities

Kasper Fugger et al. J Cell Biol. 2009.

Abstract

Homologous recombination (HR) is essential for faithful repair of DNA lesions yet must be kept in check, as unrestrained HR may compromise genome integrity and lead to premature aging or cancer. To limit unscheduled HR, cells possess DNA helicases capable of preventing excessive recombination. In this study, we show that the human Fbh1 (hFbh1) helicase accumulates at sites of DNA damage or replication stress in a manner dependent fully on its helicase activity and partially on its conserved F box. hFbh1 interacted with single-stranded DNA (ssDNA), the formation of which was required for hFbh1 recruitment to DNA lesions. Conversely, depletion of endogenous Fbh1 or ectopic expression of helicase-deficient hFbh1 attenuated ssDNA production after replication block. Although elevated levels of hFbh1 impaired Rad51 recruitment to ssDNA and suppressed HR, its small interfering RNA-mediated depletion increased the levels of chromatin-associated Rad51 and caused unscheduled sister chromatid exchange. Thus, by possessing both pro- and anti-recombinogenic potential, hFbh1 may cooperate with other DNA helicases in tightly controlling cellular HR activity.

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Figures

Figure 1.

hFbh1 accumulates at sites of DNA damage in S and G2 phase cells. (A) U2OS/GFP-hFbh1 WT cells were induced to express GFP-hFbh1 by addition of DOX for 24 h, and were subsequently left untreated or subjected to laser microirradiation, IR (6 Gy), or HU treatment. 1 h later, the cells were fixed and coimmunostained with antibodies to RPA and γ-H2AX. Magnifications of HU- and IR-treated cells are shown (red lines). (B) U2OS/GFP-hFbh1 WT cells induced to express the transgene for 24 h were subjected to IR (6 Gy) for 1 h, pulsed with BrdU for 20 min, and fixed and coimmunostained with the indicated antibodies. (C) U2OS/GFP-hFbh1 WT cells induced with DOX for 24 h were incubated in the presence of HU for the indicated times and processed for immunofluorescence as in A. Bars, 10 µm.

Figure 2.

hFbh1 interacts with ssDNA. (A) U2OS/GFP-hFbh1 WT cells were transfected with the indicated siRNAs for 24 h, induced with DOX for an additional 24 h, and exposed to IR or HU for 1 h. (top) The cells were fixed, and the GFP signal was visualized by confocal microscopy. (bottom) Immunoblot analysis of siRNA-mediated knockdown efficiency. (B) U2OS/GFP-hFbh1 WT cells were incubated in the presence of BrdU and DOX for 24 h. The cells were treated with HU for 1 h, fixed, and immunostained with BrdU antibody under native conditions. (C) EMSA is shown. 32P-labeled ssDNA or dsDNA probes were incubated with increasing amounts (200–1,000 nM) of GST-hFbh1 and subjected to native gel electrophoresis. Migration of the probe and the GST-hFbh1–ssDNA complex is indicated. Bars, 10 µm.

Figure 3.

hFbh1 acts as an anti-recombinase by displacing Rad51 from chromatin. (A) U2OS/GFP-hFbh1 WT cells were induced or not with DOX for 24 h and subjected to HU for 2 h or IR for 1 h. (left) The cells were preextracted to remove soluble proteins, fixed, and coimmunostained with RPA and Rad51 antibodies. (right) Quantification of the results of three independent experiments is shown. (B) U2OS/GFP-hFbh1 WT cells subjected to DOX and HU treatment as in A were harvested and processed for subcellular fractionation. Chromatin-enriched fractions and whole cell extracts (WCE) were immunoblotted with the indicated antibodies. Relative intensity of the Rad51 signal is indicated. (C) BJ fibroblasts were transfected with control or two independent hFbh1 siRNAs for 48 h. (top) Cells were incubated with EdU for 30 min, preextracted, fixed, and immunostained with Rad51 antibody and EdU. Two representative fields from each transfected population are shown. (bottom) The intensity of chromatin-bound Rad51 staining was quantified by image analysis software and depicted in a box plot. SID, signal-integrated density. At least 50 EdU-positive cells were analyzed in each experiment. (D) U2OS/DR-GFP cells were transfected with plasmids encoding RFP, I-SceI, and, where indicated, empty vector or Flag-hFbh1 for 48 h. Cells were processed for flow cytometric analysis of RFP and GFP, and the extent of HR was scored as the GFP/RFP ratio. The experiment was performed in triplicates. Error bars indicate the standard deviation. Bars, 10 µm.

Figure 4.

The helicase activity of hFbh1 facilitates ssDNA generation at replication blocks. (A) Schematic depiction of the hFbh1 protein, showing localization of conserved domains. Positions of residues mutated to generate hFbh1 F box (*FB) and helicase domain (*HL) mutants are indicated in red. (B) U2OS/NLS–GFP-hFbh1 cell lines were induced or not with DOX for 24 h, treated with HU for 1 h, and fixed. Where indicated, cells were preextracted before fixation. The cells were coimmunostained with the indicated antibodies. (C) U2OS/NLS–GFP-hFbh1 cell lines were treated with DOX and HU as in B and processed for subcellular fractionation. Chromatin-enriched fractions (CHR) or whole cell extracts (WCE) were immunoblotted with the indicated antibodies. (D) U2OS/GFP-hFbh1 cells were transfected with control or hFbh1 siRNA for 48 h and incubated in the presence of BrdU for an additional 24 h. Cells transfected with hFbh1 siRNA were either left uninduced or induced with DOX for the last 24 h. Cells were treated with HU for 2 h and processed for native fixation and immunostaining with BrdU antibody. BrdU staining was quantified by image analysis software and depicted in a box plot. SID, signal-integrated density. At least 1,000 cells were analyzed in each experiment. Bars, 10 µm.

Figure 5.

Depletion of hFbh1 increases SCE. (A) BJ cells were transfected with control or hFbh1 siRNAs for 48 h and subjected to SCE analysis. Metaphase chromosome spreads were prepared, and the number of SCE events was scored on a per cell basis and subjected to statistical analysis. The image shows a representative metaphase chromosome spread from hFbh1-depleted cells. The inset shows a magnification of an SCE event in the boxed region. Efficiency of hFbh1 knockdown is shown in

Fig. S3

. Bar, 10 µm. (B) Box plot showing results of the experiment in A. The increase in SCE events in hFbh1-depleted cells was reproduced in an independent experiment. At least 25 metaphase spreads were analyzed in each experiment. (C) SCE assay of cells as in B. 2.5 nM camptothecin (CPT) was present in the medium throughout the course of the experiment. (D) A hypothetical model of the pro- and anti-recombinase function of hFbh1 at stalled replication forks (left) and resected DSBs (right). See Materials and methods for further details.

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Human Fbh1 helicase contributes to genome maintenance via pro- and anti-recombinase activities - PubMed (original) (raw)