Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase

Chen, Jianhong; Morrical, Milagros D.; Donigan, Katherine A.; Weidhaas, Joanne B.; Sweasy, Joann B.; Averill, April M.; Tomczak, Jennifer A.; Morrical, Scott W.

doi:10.1093/nar/gku1337

Cited by 28 publications

(53 citation statements)

References 40 publications

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“…Fragment 1 (T1) is the N-terminal domain of RAD51, containing residues 86 and 89, which are essential for RAD51 homo-oligomerization and filament formation (31). Fragment T2 contains the Walker motifs essential for ATP-binding and nucleotide-binding (32). Fragment T3 contains the BRCA2-binding domain (30) and fragment T4 is the C-terminus of RAD51.…”

Section: Resultsmentioning

confidence: 99%

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%

A cell-penetrating antibody inhibits human RAD51 via direct binding

Turchick¹,

Hegan²,

Jensen³

et al. 2017

Nucleic Acids Research

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“…However, recent studies identified three breast cancer-associated RAD51 missense mutations, the somatic variants D149N and G151D, and the germline variant R150Q (19–21). These mutations, from different individuals, occurred in adjacent residues of a conserved Schellman loop motif that occupies a prominent position on the outer surface of the RAD51 presynaptic filament (20). The motif is distant from binding sites for DNA, ATP, BRCA2 and PALB2, but it is near a reported p53 binding site (23–25).…”

Section: Introductionmentioning

confidence: 99%

“…The motif is distant from binding sites for DNA, ATP, BRCA2 and PALB2, but it is near a reported p53 binding site (23–25). All three RAD51 mutants are proficient in DNA pairing and strand exchange, but they form presynaptic filaments with altered physical and biochemical properties (20). The R150Q and G151D variants have low catalytic efficiencies for ssDNA-stimulated ATP hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

RAD51 variant proteins from human lung and kidney tumors exhibit DNA strand exchange defects

et al. 2016

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In human cells, error-free repair of DNA double-strand breaks requires the DNA pairing and strand exchange activities of RAD51 recombinase. Activation of RAD51 recombination activities requires the assembly of RAD51 presynaptic filaments on the single-stranded DNA that forms at resected DSB ends. Mutations in proteins that control presynaptic filament assembly, such as BRCA2, and in RAD51 itself, are associated with human breast cancer. Here we describe the properties of two mutations in RAD51 protein that derive from human lung and kidney tumors, respectively. Sequence variants Q268P and Q272L both map to the DNA binding loop 2 (L2) region of RAD51, a motif that is involved in DNA binding and in the allosteric activation of ATP hydrolysis and DNA strand exchange activities. Both mutations alter the thermal stability, DNA binding, and ATPase properties of RAD51, however both variants retain intrinsic DNA strand exchange activity towards oligonucleotide substrates under optimized conditions. In contrast, both Q268P and Q272L variants exhibit drastically reduced DNA strand exchange activity in reaction mixtures containing long homologous ssDNA and dsDNA substrates and human RPA protein. Mixtures of wild-type and variant proteins also exhibit reduced DNA strand exchange activity, suggesting that heterozygous mutations could negatively affect DNA recombination and repair processes in vivo. Together, the findings of this study suggest that hypomorphic missense mutations in RAD51 protein could be drivers of genomic instability in cancer cells, and thereby contribute to the etiology of metastatic disease.

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“…1C) the greatest similarity in the central and carboxyl terminal regions, but their N-terminal regions were less well aligned. Interestingly, GdDMC1A has an extended amino terminal region and lacks the recently described Schellman loop [30] (Fig. 1B), while GdDMC1B conserves this motif.…”

Section: Gddmc1a and Gddmc1b Possess Conserved Recombinase Domainsmentioning

confidence: 67%

“…The specific functions of each GdDMC1 might depend on the possible interactions in this region. In addition to the conventional motifs identified in giardial recombinases, a new motif was recently identified: the Schellman loop [30]. The Schellman loop seems to be conserved among Rad51 proteins from different species, and it could assist in interactions with solvents, ions or protein partners.…”

Section: Discussionmentioning

confidence: 99%