A chemical compound that stimulates the human homologous recombination protein RAD51.
RAD51 and other members of the RecA family of strand exchange proteins assemble on ssDNA to form presynaptic filaments, which carry out the central steps of homologous recombination. A microplate-based assay was developed for high-throughput measurement of hRAD51 filament formation on ssDNA. With this method, a 10,000 compound library was screened, leading to the identification of a small molecule (RS-1) that enhances hRAD51 binding in a wide range of biochemical conditions. Salt titration experiments showed that RS-1 can enhance filament stability. Ultrastructural analysis of filaments formed on ssDNA showed that RS-1 can increase both protein-DNA complex lengths and the pitch of helical filament turns. RS-1 stimulated hRAD51-mediated homologous strand assimilation (D-loop) activity by at least 5- to 11-fold, depending on the condition. This D-loop stimulation occurred even in the presence of Ca(2+) or adenylyl-imidodiphosphate, indicating that the mechanism of stimulation was distinct from that conferred by Ca(2+) and/or inhibition of ATPase. No D-loop activity was observed in the absence of a nucleotide triphosphate cofactor, indicating that the compound does not substitute for this requirement. These results indicate that RS-1 enhances the homologous recombination activity of hRAD51 by promoting the formation of active presynaptic filaments. Cell survival assays in normal neonatal human dermal fibroblasts demonstrated that RS-1 promotes a dose-dependent resistance to the cross-linking chemotherapeutic drug cisplatin. Given that RAD51-dependent recombination is a major determinant of cisplatin resistance, RS-1 seems to function in vivo to stimulate homologous recombination repair proficiency. RS-1 has many potential applications in both research and medical settings.
Department of Radiation and Cellular Oncology, University of Chicago, Chicago, IL 60637, USA.
Krishanthi
K
Jayathilaka
Sean D
SD
Sheridan
Tyler D
TD
Bold
Katarzyna
K
Bochenska
Hillary L
HL
Logan
Ralph R
RR
Weichselbaum
Douglas K
DK
Bishop
Philip P
PP
Connell
D001549:Benzamides / Q000494:pharmacology*; D002470:Cell Survival; D002945:Cisplatin; D004277:DNA, Single-Stranded / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D004795:Enzyme Stability / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007231:Infant, Newborn; D008024:Ligands; D051135:Rad51 Recombinase / Q000187:drug effects*; D054852:Small Molecule Libraries / Q000494:pharmacology*; D013449:Sulfonamides / Q000494:pharmacology*
C000605335:3-((benzylamino)sulfonyl)-4-bromo-N-(4-bromophenyl)benzamide; C000592317:3-(N-benzylsulfamoyl)-4-bromo-N-(4-bromophenyl)benzamide; D001549:Benzamides; D004277:DNA, Single-Stranded; D008024:Ligands; D054852:Small Molecule Libraries; D013449:Sulfonamides; D051135:Rad51 Recombinase; D002945:Cisplatin