Germline RBBP8 variants associated with early-onset breast cancer compromise replication fork stability
Reihaneh Zarrizi, Martin R. Higgs, Karolin Voßgröne, Maria Rossing, Birgitte Bertelsen, Muthiah Bose, Arne Nedergaard Kousholt, Heike Rösner, the COMPLEXO Network, Bent Ejlertsen, Grant S. Stewart, Finn Cilius Nielsen, Claus S. Sørensen
Reihaneh Zarrizi, Martin R. Higgs, Karolin Voßgröne, Maria Rossing, Birgitte Bertelsen, Muthiah Bose, Arne Nedergaard Kousholt, Heike Rösner, the COMPLEXO Network, Bent Ejlertsen, Grant S. Stewart, Finn Cilius Nielsen, Claus S. Sørensen
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Research Article Cell biology Genetics

Germline RBBP8 variants associated with early-onset breast cancer compromise replication fork stability

Abstract

Haploinsufficiency of factors governing genome stability underlies hereditary breast and ovarian cancer. One significant pathway that is disabled as a result is homologous recombination repair (HRR). With the aim of identifying new candidate genes, we examined early-onset breast cancer patients negative for BRCA1 and BRCA2 pathogenic variants. Here, we focused on CtIP (RBBP8 gene), which mediates HRR through the end resection of DNA double-strand breaks (DSBs). Notably, these patients exhibited a number of rare germline RBBP8 variants. Functional analysis revealed that these variants did not affect DNA DSB end resection efficiency. However, expression of a subset of variants led to deleterious nucleolytic degradation of stalled DNA replication forks in a manner similar to that of cells lacking BRCA1 or BRCA2. In contrast to BRCA1 and BRCA2, CtIP deficiency promoted the helicase-driven destabilization of RAD51 nucleofilaments at damaged DNA replication forks. Taken together, our work identifies CtIP as a critical regulator of DNA replication fork integrity, which, when compromised, may predispose to the development of early-onset breast cancer.

Authors

Reihaneh Zarrizi, Martin R. Higgs, Karolin Voßgröne, Maria Rossing, Birgitte Bertelsen, Muthiah Bose, Arne Nedergaard Kousholt, Heike Rösner, the COMPLEXO Network, Bent Ejlertsen, Grant S. Stewart, Finn Cilius Nielsen, Claus S. Sørensen

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Figure 4

CtIP promotes replication fork protection through FBH1.

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CtIP promotes replication fork protection through FBH1. (A) Experimental...
(A) Experimental scheme of dual labeling of DNA fibers in doxycycline-inducible U-2-OS cells stably expressing the siRNA-resistant full-length WT, E804del, or ΔC CtIP. Cells were sequentially pulse-labeled with CldU and IdU, then treated with 4 mM HU for 5 hours. (B and C) Loss of CtIP results in replication fork instability in response to replication stress. Doxycycline-inducible U-2-OS cells were transfected with CtIP siRNA, and 24 hours later, induced with doxycycline for 24 hours. IdU/CldU rations are given. (D and E) MCF7 cells were transfected with the indicated siRNAs. Cells were pulsed with 10 µM EdU for 20 minutes before addition of 4 mM HU. Cells in S-phase (EdU+) at the time of HU treatments were Click-IT-labeled with an Alexa Fluor 594 azide. (D) Representative images of RAD51 foci. (E) RAD51 foci in EdU+ cells were enumerated using ImageJ/Fiji. The displayed data represent 3 independent biological replicates, and n = 224 nuclei were analyzed per sample. Holm-corrected multiple testing was performed of ranked data fitted by a linear mixed model. ***P < 0.0001. (F) U-2-OS cells were transfected with the indicated siRNAs and exposed to 4 mM HU for 5 hours. IdU/CldU ratios are given. (G) U-2-OS cells were transfected with the indicated siRNAs and exposed to 4 mM HU for 5 hours. IdU/CldU ratios are given. (H) U-2-OS cells were transfected with the indicated siRNAs and exposed to 4 mM HU for 5 hours. IdU/CldU ratios are given. (I) Schematic model for the role of CtIP at stalled forks. CtIP regulates RAD51 stability at stalled forks, counteracting the dissolution of the RAD51 filament by FBH1. Loss of CtIP leads to DNA damage accumulation and enhanced chromosomal instability.