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 Y.; Luong, Thong; 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.

doi:10.1016/j.dnarep.2019.02.008

Cited by 18 publications

(14 citation statements)

References 57 publications

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“…As a proof of concept, we demonstrated the ease with which these mutant lines can be used to test the HR proficiency of RAD51 paralog variants of clinical interest in a small pilot screen with RAD51B . In addition, we have recently utilized these cell lines to perform similar HR analysis with RAD51C and RAD51D patient-derived reversions and mutations [102,103]. This kind of analysis will be particularly useful for investigation of the numerous RAD51 paralog variants of unknown significance found in patient cancer cells which may in the long-term help optimize personalized cancer therapies.…”

Section: Discussionmentioning

confidence: 99%

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells

et al. 2019

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Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib, with the weakest phenotypes observed in RAD51B-deficient cells. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.

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Section: Discussionmentioning

confidence: 99%

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells

et al. 2019

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“…Mutating the highly conserved lysine (K114) in the Walker A domain to glutamine (Q) in RAD51B did not affect its complementing activity in HR assays suggesting that either this lysine residue is not as important as the other conserved residues in this domain or that the substitution of the lysine to glutamine would be tolerated by cells. In addition, we have recently utilized these cell lines to perform similar HR analysis with RAD51C and RAD51D patient-derived reversions and mutations, respectively (Baldock et al 2019;Kondrashova et al 2017). This kind of analysis will be particularly useful for investigation of the numerous RAD51 paralog variants of unknown significance found in patient cancer cells which may in the long-term help optimize personalized cancer therapies.…”

Section: Discussionmentioning

confidence: 99%

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells

Garcin

Gon

Prakash

et al. 2019

Preprint

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Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hypersensitivity to mitomycin C and olaparib. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.

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“…The following are available online at , Figure S1: Workflow of the minigene protocol, Figure S2: Insert sequence of minigene mgR51D_ex2–9, Figure S3: Agarose gel (1.0%) electrophoresis of the splicing assays of eight splice-site variants in MDA-MB-231 and MCF-7 cells, Figure S4: Schematic representation of the most common aberrant transcripts, Figure S5: Fluorescent fragment analysis of other exon 3 microdeletions and variants, Figure S6: Mapping human RAD51D regions critical to protein function, Figure S7: Mapping of 9 RAD51D C-terminal β-strands, Figure S8: PS3/BS3 annotation of RAD51D -altered and canonical transcripts, Table S1: Mutagenesis primers for RAD51D variants and microdeletions, Table S2: Bioinformatics analysis of RAD51D variants with Max Ent Score, Table S3: RNA and protein HGVS annotations according to transcript ENST00000345365.10, Table S4: Functional mapping of ESEs by exonic microdeletions, Table S5: Accuracy of bioinformatics predictions, Table S6: Sensitivity and specificity of the splicing programs, Supplementary Methods: ACMG/AMP-like classification of 37 RAD51D variants based on PS3/BS3 functional evidence. References [ 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 ] are cited in Supplementary Materials.…”

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RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants

Bueno-Martínez

Sanoguera-Miralles

Valenzuela-Palomo

et al. 2021

Cancers

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RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2–9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (<3.4%) of the full-length (FL) transcript. In addition, ESE elements of the alternative exon 3 were mapped by testing four overlapping exonic microdeletions (≥30-bp), revealing an ESE-rich interval (c.202_235del) with critical sequences for exon 3 recognition that might have been affected by germline variants. Next, 26 BRIDGES variants and 16 artificial exon 3 single-nucleotide substitutions were also assayed. Thirty variants impaired splicing with variable amounts (0–65.1%) of the FL transcript, although only c.202G > A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T > C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation.

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

Cited by 18 publications

References 57 publications

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells

RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants

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