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Cancer-associated mutations in the iron-sulfur domain of FANCJ affect G-quadruplex metabolism - PubMed

  • ️Wed Jan 01 2020

Cancer-associated mutations in the iron-sulfur domain of FANCJ affect G-quadruplex metabolism

Diana C Odermatt et al. PLoS Genet. 2020.

Abstract

FANCJ/BRIP1 is an iron-sulfur (FeS) cluster-binding DNA helicase involved in DNA inter-strand cross-link (ICL) repair and G-quadruplex (G4) metabolism. Mutations in FANCJ are associated with Fanconi anemia and an increased risk for developing breast and ovarian cancer. Several cancer-associated mutations are located in the FeS domain of FANCJ, but how they affect FeS cluster binding and/or FANCJ activity has remained mostly unclear. Here we show that the FeS cluster is indispensable for FANCJ's ability to unwind DNA substrates in vitro and to provide cellular resistance to agents that induce ICLs. Moreover, we find that FANCJ requires an intact FeS cluster for its ability to unfold G4 structures on the DNA template in a primer extension assay with the lagging-strand DNA polymerase delta. Surprisingly, however, FANCJ variants that are unable to bind an FeS cluster and to unwind DNA in vitro can partially suppress the formation of replisome-associated G4 structures that we observe in a FANCJ knock-out cell line. This may suggest a partially retained cellular activity of FANCJ variants with alterations in the FeS domain. On the other hand, FANCJ knock-out cells expressing FeS cluster-deficient variants display a similar-enhanced-sensitivity towards pyridostatin (PDS) and CX-5461, two agents that stabilise G4 structures, as FANCJ knock-out cells. Mutations in FANCJ that abolish FeS cluster binding may hence be predictive of an increased cellular sensitivity towards G4-stabilising agents.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. FANCJ coordinates an FeS cluster that is essential for MMC resistance.

(A) Alignment of FANCJ sequences from a variety of species. FeS cluster-coordinating cysteines are depicted in yellow, disease-associated and rationally designed alterations are highlighted in colour and annotated. (B) Radioactive iron-55 incorporation by FANCJ variants, as measured by liquid scintillation counting. Levels are expressed as % iron incorporation, with wild-type levels set to 100%. The non-FeS protein PCNA was used as a negative control. Error bars depict standard deviations from three independent experiments. Statistical analysis: ordinary one-way ANOVA (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; ns, non-significant). (C) MMC sensitivity of FANCJ HeLa FIT knock-out cells (FJ–/–) complemented with different FANCJ constructs. Graph depicts mean values of three independent experiments. For raw values, standard deviations and statistical analysis see S1 Table. WT, wild-type; KR, K52R; RQ, R279Q; CS, C283S; CH, C283H; CR, C283R; MI, M299I; LF, L340F; AP, A349P. See also S1 Fig.

Fig 2
Fig 2. The FeS cluster in FANCJ is indispensable for helicase activity.

(A) InstantBlue stained SDS-PAGE gel of purified N-terminally Flag-tagged FANCJ variants. Asterisks mark contaminants. (B) ATP hydrolysis of FANCJ variants in the presence of an oligonucleotide-based Y-structure (Y) or D-loop (D) substrate, as measured by the release of inorganic phosphate from radio-labelled γ-32P-ATP in thin-layer chromatography. Activity is depicted as % of hydrolysed ATP, with background activity in the absence of DNA subtracted. Error bars depict standard deviations from three independent experiments. Statistical analysis: ordinary one-way ANOVA (****, p < 0.0001; **, p < 0.01; *, p < 0.1; ns, non-significant). (C) Graphical representation of DNA unwinding of a D-loop substrate with increasing concentrations of FANCJ variants. In the graph, mean values and standard deviations of three independent experiments are depicted. WT, wild-type; KR, K52R; CS, C283S; CR, C283R; MI, M299I; LF, L340F; AP, A349P. See also S2 Fig.

Fig 3
Fig 3. FANCJ can resolve G4 structures ahead of Pol δ in vitro.

(A) Scheme of primer-template substrate with a sequence forming a parallel G4 structure on the template strand ahead of the primer that can be extended by Pol δ. Asterisk indicates 5´-FAM label. Numbers indicate lengths of primers and gaps in nucleotides (nt). (B) Time-resolved primer extension assay with 10 nM of Pol δ alone (–) and in the presence of 35 nM FANCJ wild-type or variants. Numbers indicate the mean percentage of full extension and standard deviations from two independent experiments. (C,D) Primer extension assay with 10 nM of Pol δ and increasing amounts of different FANCJ variants (C) and quantification (D). In the graph, mean values and standard deviations of two independent experiments are depicted. N+12 denotes position of G4 block relative to 3´-end of primer. S, DNA substrate without protein; WT, wild-type; KR, K52R; CS, C283S; CR, C283R; MI, M299I; LF, L340F; AP, A349P.

Fig 4
Fig 4. The FeS cluster is required to efficiently prevent replisome-associated G4 structures.

(A) Representative SMLM images labelled for nascent DNA (red), G4 structures (blue), and MCM (green) to detect replisome-associated G4 structures. Scale bar, 2 μm. (B) Densities of replisome-associated G4 structures in FANCJ knock-out cells (KO) complemented with the indicated variants. Each cell line was either non-treated (NT) or treated for 1h with 20 μM PDS. Individual data point represents result from one nucleus. Black horizontal line and error bars indicate mean ± standard deviation. Values on graph indicate p-values of unpaired two-sample t-tests between NT and PDS-treated cells. Note that for technical reasons not all cell lines were treated in parallel, and, hence, absolute values between cell lines were not compared. For raw values and analysis see S2 Table. (C) Averaged fold change of the densities of replisome-associated G4 structures in PDS-treated compared to non-treated cells. Bold horizontal lines represent the ratio between the mean values of PDS-treated and NT cells from (B), and the error bars indicate the propagated standard errors of the mean. (D) Sensitivity of FANCJ HeLa FIT knock-out cells (FJ–/–) complemented with different FANCJ constructs to PDS treatment. In the graph, the mean values of three independent experiments are depicted. For raw values, standard deviations and statistical analysis see S3 Table. WT, wild-type; KR, K52R; CS, C283S; CR, C283R; MI, M299I; LF, L340F; AP, A349P.

Fig 5
Fig 5. FANCJ knock-out cells display enhanced sensitivity to the G4-stabilising agent CX-5461.

(A) Sensitivity of FANCJ HeLa FIT knock-out cells (FJ–/–) complemented with different FANCJ constructs to CX-5461 treatment. In the graphs, the mean values of three independent experiments are depicted. For raw values, standard deviations and statistical analysis see S4 Table. CX, CX-5461; WT, wild-type; KR, K52R; CS, C283S; CR, C283R; MI, M299I; LF, L340F; AP, A349P. (B) Model showing wild-type FANCJ able to resolve G4 structures on the templating strand during DNA replication, which would allow continuous DNA synthesis (upper panel). In contrast, the inability of FeS cluster-deficient FANCJ to efficiently resolve G4 structures would lead to polymerase stalling and persistent replisome-associated G4 structures (lower panel).

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