Distinct binding of BRCA2 BRC repeats to RAD51 generates differential DNA damage sensitivity

Chatterjee, Gouri; Jiménez-Sáinz, Judit; Presti, Thomas; Nguyen, Tiffany; Jensen, Ryan B.

doi:10.1093/nar/gkw242

Cited by 57 publications

(100 citation statements)

References 61 publications

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“…Figure 2A documents that all five scFv proteins are expressed upon transient transfection of the expression vectors into primary human skin fibroblasts, and further demonstrates primarily cytoplasmic localization. We then interrogated repair of an I-SceI nuclease-induced DSB within a luciferase reporter gene (29) in the presence of each expressed scFv. A schematic of the luciferase reporter construct used to assay HDR is provided in Figure 2B.…”

Section: Resultsmentioning

confidence: 99%

“…Because the D31N mutation appeared to alter the interaction of the 3E10 scFv with cellular RAD51, we further evaluated the relative binding affinities of purified WT and D31N scFv proteins for purified RAD51 in vitro (29). Purified WT and D31N scFv proteins at a fixed concentration (400 ng in a final volume of 50 μl; 72.7 nM) were incubated with increasing amounts of purified RAD51 (100, 200, 800, 1600 ng; 54, 108, 432 and 864 nM, respectively), and the interacting complex was pulled down via the 2XMBP affinity tag on the scFvs using amylose resin.…”

Section: Resultsmentioning

confidence: 99%

“…It is thought that under cellular stress and DNA damage, RAD51 is transported to the nucleus and loaded onto DNA by BRCA2 (29). RAD51C, RAD52 and BRCA2 have all been suggested to play a role in the translocation of RAD51 from the cytoplasm to the nucleus although the exact mechanism is yet to be determined (29,33,34). Therefore, we hypothesize that, since the 2XMBP-scFvs are primarily localized in the cytoplasm, the interaction between RAD51 and the 3E10 scFvs likely occurs in the cytoplasm.…”

Section: Resultsmentioning

confidence: 99%

“…BRCA2 and PTEN are both known to play roles in DSB repair, and cells deficient in either of these proteins have been reported to have elevated levels of unrepaired DNA damage (4,5,29,35,36). Previous publications have reported that 3E10 is synthetically lethal with BRCA2-deficiency as well as PTEN-deficiency (26,37).…”

Section: Resultsmentioning

confidence: 99%

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A cell-penetrating antibody inhibits human RAD51 via direct binding

Turchick¹,

Hegan²,

Jensen³

et al. 2017

Nucleic Acids Research

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RAD51, a key factor in homology-directed repair (HDR), has long been considered an attractive target for cancer therapy, but few specific inhibitors have been found. A cell-penetrating, anti-DNA, lupus autoantibody, 3E10, was previously shown to inhibit HDR, sensitize tumors to radiation, and mediate synthetic lethal killing of BRCA2-deficient cancer cells, effects that were initially attributed to its affinity for DNA. However, as the molecular basis for its ability to inhibit DNA repair, we report that 3E10 directly binds to the N-terminus of RAD51, sequesters RAD51 in the cytoplasm, and impedes RAD51 binding to DNA. Further, we generate separation-of-function mutations in the complementarity-determining regions of 3E10 revealing that inhibition of HDR tracks with binding to RAD51 but not to DNA, whereas cell penetration is linked to DNA binding. The consequences of these mutations on putative 3E10 interactions with RAD51 and DNA are correlated with in silico molecular modeling. Taken together, the results identify 3E10 as a novel inhibitor of RAD51 by direct binding, accounting for its ability to suppress HDR and providing the molecular basis to guide pre-clinical development of 3E10 as an anti-cancer agent.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

A cell-penetrating antibody inhibits human RAD51 via direct binding

Turchick¹,

Hegan²,

Jensen³

et al. 2017

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…The HR luciferase reporter has been previously reported(28, 61) and was generated by cloning an inactivating I-SceI recognition site into the BstBI site 56 amino acids into the firefly luciferase gene in the gWIZ.Luciferase vector (Gelantis), and cloning a promoterless copy of the firefly luciferase open reading frame 700 base pairs downstream in reverse orientation as a donor template for HR. A DSB in the firefly luciferase gene was induced by I-SceI digestion and confirmed by electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity

et al. 2017

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2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations, whereas the latter is produced under pathologic processes such as hypoxia. Here, we report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. This “BRCAness” phenotype of IDH mutant cells can be completely reversed by treatment with small molecule inhibitors of the mutant IDH1 enzyme, and, conversely, it can be entirely recapitulated by treatment with either 2HG enantiomer alone in cells with intact IDH1/2 proteins. We demonstrate IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR-deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

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