title: RAD51 variant proteins from human lung and kidney tumors exhibit DNA strand exchange defects.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405, United States.
authors.forename: Michelle C
authors.initials: MC
authors.lastname: Silva
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405, United States.
authors.forename: Milagros D
authors.initials: MD
authors.lastname: Morrical
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405, United States.
authors.forename: Katie E
authors.initials: KE
authors.lastname: Bryan
authors.affiliation: Department of Microbiology & Molecular Genetics, University of Vermont College of Medicine, Burlington, VT, 05405 United States.
authors.forename: April M
authors.initials: AM
authors.lastname: Averill
authors.affiliation: Department of Microbiology & Molecular Genetics, University of Vermont College of Medicine, Burlington, VT, 05405 United States.
authors.forename: Julie
authors.initials: J
authors.lastname: Dragon
authors.affiliation: Department of Microbiology & Molecular Genetics, University of Vermont College of Medicine, Burlington, VT, 05405 United States; University of Vermont Cancer Center, University of Vermont College of Medicine, Burlington, VT 05405 United States.
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Bond
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT 05405, United States; Department of Microbiology & Molecular Genetics, University of Vermont College of Medicine, Burlington, VT, 05405 United States; University of Vermont Cancer Center, University of Vermont College of Medicine, Burlington, VT 05405 United States. Electronic address: smorrica@uvm.edu.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Morrical
mesh_terms: D004247:DNA / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000378:metabolism*; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism*; D008875:Middle Aged; D008958:Models, Molecular; D020125:Mutation, Missense; D011487:Protein Conformation; D055550:Protein Stability; D051135:Rad51 Recombinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013696:Temperature
chemical_list: D004247:DNA; D051135:Rad51 Recombinase
keywords: Cancer; Homology-directed repair; Presynaptic filament; RAD51; Recombination; Variant