RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination

Robert A Baldock, Catherine A Pressimone, Jared M Baird, Anton Khodakov, Thong T Luong, McKenzie K Grundy, Chelsea M Smith, Yoav Karpenshif, Dominique S Bratton-Palmer, Rohit Prakash, Maria Jasin, Edwige B Garcin, Stéphanie Gon, Mauro Modesti, Kara A Bernstein

Research output: Contribution to journalArticle

Abstract

The proficiency of cancer cells to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) is a key determinant in predicting response to targeted therapies such as PARP inhibitors. The RAD51 paralogs work as multimeric complexes and act downstream of BRCA1 to facilitate HR. Numerous epidemiological studies have linked RAD51 paralog mutations with hereditary cancer predisposition. Despite their substantial links to cancer, RAD51 paralog HR function has remained elusive. Here we identify isoform 1 as the functional isoform of RAD51D, whereas isoform 4 which has a large N-terminal deletion (including the Walker A motif), and isoform 6 which includes an alternate exon in the N-terminus, are non-functional. To determine the importance of this N-terminal region, we investigated the impact of cancer-associated mutations and SNPs in this variable RAD51D N-terminal region using yeast-2-hybrid and yeast-3-hybrid assays to screen for altered protein-protein interactions. We identified two cancer-associated mutations close to or within the Walker A motif (G96C and G107 V, respectively) that independently disrupt RAD51D interaction with XRCC2. We validated our yeast interaction data in human U2OS cells by co-immunoprecipitation and determined the impact of these mutations on HR-proficiency using a sister chromatid recombination reporter assay in a RAD51D knock-out cell line. Our investigation reveals that the interaction of RAD51D with XRCC2 is required for DSB repair. By characterizing the impact of cancer-associated mutations on RAD51D interactions, we aim to develop predictive models for therapeutic sensitivity and resistance in patients who harbor similar mutations in RAD51D.

Original languageEnglish
Pages (from-to)99-107
Number of pages9
JournalDNA Repair
Volume76
DOIs
Publication statusPublished - Apr 2019
Externally publishedYes

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Homologous Recombination
Protein Isoforms
Yeast
Mutation
Assays
Neoplasms
Repair
Yeasts
Cells
Ports and harbors
Exons
Proteins
Chromatids
Double-Stranded DNA Breaks
Immunoprecipitation
Genetic Recombination
Single Nucleotide Polymorphism
Siblings
Epidemiologic Studies
DNA

Cite this

Baldock, Robert A ; Pressimone, Catherine A ; Baird, Jared M ; Khodakov, Anton ; Luong, Thong T ; Grundy, McKenzie K ; Smith, Chelsea M ; Karpenshif, Yoav ; Bratton-Palmer, Dominique S ; Prakash, Rohit ; Jasin, Maria ; Garcin, Edwige B ; Gon, Stéphanie ; Modesti, Mauro ; Bernstein, Kara A. / RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination. In: DNA Repair. 2019 ; Vol. 76. pp. 99-107.
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abstract = "The proficiency of cancer cells to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) is a key determinant in predicting response to targeted therapies such as PARP inhibitors. The RAD51 paralogs work as multimeric complexes and act downstream of BRCA1 to facilitate HR. Numerous epidemiological studies have linked RAD51 paralog mutations with hereditary cancer predisposition. Despite their substantial links to cancer, RAD51 paralog HR function has remained elusive. Here we identify isoform 1 as the functional isoform of RAD51D, whereas isoform 4 which has a large N-terminal deletion (including the Walker A motif), and isoform 6 which includes an alternate exon in the N-terminus, are non-functional. To determine the importance of this N-terminal region, we investigated the impact of cancer-associated mutations and SNPs in this variable RAD51D N-terminal region using yeast-2-hybrid and yeast-3-hybrid assays to screen for altered protein-protein interactions. We identified two cancer-associated mutations close to or within the Walker A motif (G96C and G107 V, respectively) that independently disrupt RAD51D interaction with XRCC2. We validated our yeast interaction data in human U2OS cells by co-immunoprecipitation and determined the impact of these mutations on HR-proficiency using a sister chromatid recombination reporter assay in a RAD51D knock-out cell line. Our investigation reveals that the interaction of RAD51D with XRCC2 is required for DSB repair. By characterizing the impact of cancer-associated mutations on RAD51D interactions, we aim to develop predictive models for therapeutic sensitivity and resistance in patients who harbor similar mutations in RAD51D.",
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Baldock, RA, Pressimone, CA, Baird, JM, Khodakov, A, Luong, TT, Grundy, MK, Smith, CM, Karpenshif, Y, Bratton-Palmer, DS, Prakash, R, Jasin, M, Garcin, EB, Gon, S, Modesti, M & Bernstein, KA 2019, 'RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination' DNA Repair, vol. 76, pp. 99-107. https://doi.org/10.1016/j.dnarep.2019.02.008

RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination. / Baldock, Robert A; Pressimone, Catherine A; Baird, Jared M; Khodakov, Anton; Luong, Thong T; Grundy, McKenzie K; Smith, Chelsea M; Karpenshif, Yoav; Bratton-Palmer, Dominique S; Prakash, Rohit; Jasin, Maria; Garcin, Edwige B; Gon, Stéphanie; Modesti, Mauro; Bernstein, Kara A.

In: DNA Repair, Vol. 76, 04.2019, p. 99-107.

Research output: Contribution to journalArticle

TY - JOUR

T1 - RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination

AU - Baldock, Robert A

AU - Pressimone, Catherine A

AU - Baird, Jared M

AU - Khodakov, Anton

AU - Luong, Thong T

AU - Grundy, McKenzie K

AU - Smith, Chelsea M

AU - Karpenshif, Yoav

AU - Bratton-Palmer, Dominique S

AU - Prakash, Rohit

AU - Jasin, Maria

AU - Garcin, Edwige B

AU - Gon, Stéphanie

AU - Modesti, Mauro

AU - Bernstein, Kara A

N1 - Copyright © 2019 Elsevier B.V. All rights reserved.

PY - 2019/4

Y1 - 2019/4

N2 - The proficiency of cancer cells to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) is a key determinant in predicting response to targeted therapies such as PARP inhibitors. The RAD51 paralogs work as multimeric complexes and act downstream of BRCA1 to facilitate HR. Numerous epidemiological studies have linked RAD51 paralog mutations with hereditary cancer predisposition. Despite their substantial links to cancer, RAD51 paralog HR function has remained elusive. Here we identify isoform 1 as the functional isoform of RAD51D, whereas isoform 4 which has a large N-terminal deletion (including the Walker A motif), and isoform 6 which includes an alternate exon in the N-terminus, are non-functional. To determine the importance of this N-terminal region, we investigated the impact of cancer-associated mutations and SNPs in this variable RAD51D N-terminal region using yeast-2-hybrid and yeast-3-hybrid assays to screen for altered protein-protein interactions. We identified two cancer-associated mutations close to or within the Walker A motif (G96C and G107 V, respectively) that independently disrupt RAD51D interaction with XRCC2. We validated our yeast interaction data in human U2OS cells by co-immunoprecipitation and determined the impact of these mutations on HR-proficiency using a sister chromatid recombination reporter assay in a RAD51D knock-out cell line. Our investigation reveals that the interaction of RAD51D with XRCC2 is required for DSB repair. By characterizing the impact of cancer-associated mutations on RAD51D interactions, we aim to develop predictive models for therapeutic sensitivity and resistance in patients who harbor similar mutations in RAD51D.

AB - The proficiency of cancer cells to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) is a key determinant in predicting response to targeted therapies such as PARP inhibitors. The RAD51 paralogs work as multimeric complexes and act downstream of BRCA1 to facilitate HR. Numerous epidemiological studies have linked RAD51 paralog mutations with hereditary cancer predisposition. Despite their substantial links to cancer, RAD51 paralog HR function has remained elusive. Here we identify isoform 1 as the functional isoform of RAD51D, whereas isoform 4 which has a large N-terminal deletion (including the Walker A motif), and isoform 6 which includes an alternate exon in the N-terminus, are non-functional. To determine the importance of this N-terminal region, we investigated the impact of cancer-associated mutations and SNPs in this variable RAD51D N-terminal region using yeast-2-hybrid and yeast-3-hybrid assays to screen for altered protein-protein interactions. We identified two cancer-associated mutations close to or within the Walker A motif (G96C and G107 V, respectively) that independently disrupt RAD51D interaction with XRCC2. We validated our yeast interaction data in human U2OS cells by co-immunoprecipitation and determined the impact of these mutations on HR-proficiency using a sister chromatid recombination reporter assay in a RAD51D knock-out cell line. Our investigation reveals that the interaction of RAD51D with XRCC2 is required for DSB repair. By characterizing the impact of cancer-associated mutations on RAD51D interactions, we aim to develop predictive models for therapeutic sensitivity and resistance in patients who harbor similar mutations in RAD51D.

U2 - 10.1016/j.dnarep.2019.02.008

DO - 10.1016/j.dnarep.2019.02.008

M3 - Article

VL - 76

SP - 99

EP - 107

JO - DNA Repair

JF - DNA Repair

SN - 1568-7864

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