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Germline RBBP8 variants associated with early-onset breast cancer compromise replication fork stability
Reihaneh Zarrizi, … , Finn Cilius Nielsen, Claus S. Sørensen
Reihaneh Zarrizi, … , Finn Cilius Nielsen, Claus S. Sørensen
Published May 7, 2020
Citation Information: J Clin Invest. 2020;130(8):4069-4080. https://doi.org/10.1172/JCI127521.
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Research Article Cell biology Genetics

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

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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 3

CtIP prevents ssDNA accumulation after replication stress.

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CtIP prevents ssDNA accumulation after replication stress.
(A) Represent...
(A) Representative images displaying RPA in HU-treated EdU+ cells. Scale bars: 20 μm. (B) MCF7 cells were transfected with the indicated siRNA, and 24 hours later, cells were transfected with WT or its mutated CtIP variants. Afterward, 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 treatment were Click-iT–labeled (Thermo Fisher Scientific) with an Alexa Fluor 594 azide, and RPA intensity in EdU+ cells was enumerated using ImageJ/Fiji. The displayed data represent 3 independent biological replicates, and n ≥ 174 nuclei were analyzed per sample. Holm-corrected multiple testing was performed of ranked data fitted by a linear mixed model, comparing all CtIP variants with WT-CtIP-GFP. ***P < 0.0001. (C) Representative images displaying RAD51 in HU-treated EdU+ cells. Scale bars: 20 μm. (D) MCF7 cells were transfected with the indicated siRNA, and 24 hours later, cells were transfected with WT or mutated CtIP variants. Afterward, 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 treatment were Click-IT–labeled with an Alexa Fluor 594 azide, and RAD51 foci in EdU+ cells were enumerated using ImageJ/Fiji. The displayed data represent 3 independent biological replicates, and n ≥ 207 nuclei were analyzed per sample. Holm-corrected multiple testing was performed of ranked data fitted by a linear mixed model, comparing all CtIP variants with WT-CtIP-GFP.

Copyright © 2022 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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