NUCKS1 promotes RAD54 activity in homologous recombination DNA repair

Abstract

NUCKS1 (nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate 1) is a chromatin-associated, vertebrate-specific, and multifunctional protein with a role in DNA damage signaling and repair. Previously, we have shown that NUCKS1 helps maintain homologous recombination (HR) DNA repair in human cells and functions as a tumor suppressor in mice. However, the mechanisms by which NUCKS1 positively impacts these processes had remained unclear. Here, we show that NUCKS1 physically and functionally interacts with the DNA motor protein RAD54. Upon exposure of human cells to DNA-damaging agents, NUCKS1 controls the resolution of RAD54 foci. In unperturbed cells, NUCKS1 prevents RAD54’s inappropriate engagement with RAD51AP1. In vitro, NUCKS1 stimulates the ATPase activity of RAD54 and the RAD51–RAD54-mediated strand invasion step during displacement loop formation. Taken together, our data demonstrate that the NUCKS1 protein is an important new regulator of the spatiotemporal events in HR.

RAD51AP1 mediates RAD51 activity through nucleosome interaction

“…The purification procedures for full-length human RAD51AP1, the RAD51AP1 fragments and mutants, and for human RAD54, RAD51 and NAP1 have been described elsewhere ( 5 , 6 , 11 , 24 , 41 , 42 ). Purified human histone octamer was purchased from The Histone Source at Colorado State University ( https://www.histonesource.com/ ) and purified as described ( 43 ).…”

“…For purification of the RAD51AP1-N88 protein, E. coli Rosetta cells harboring the plasmid were grown and induced for protein expression as described earlier ( 42 ). RAD51AP1-N88 was purified by tandem purification using Ni-NTA resin (Thermo Scientific) first, as described ( 42 ).…”

RAD51AP1 mediates RAD51 activity through nucleosome interaction

“…The purification procedures for full-length human RAD51AP1, the RAD51AP1 fragments and mutants, and for human RAD54, RAD51 and NAP1 have been described elsewhere (5,6,11,24,41,42). Purified human histone octamer was purchased from The Histone Source at Colorado State University (https://www.histonesource.com/)and purified as described (43).…”

“…For purification of the RAD51AP1-N88 protein, E. coli Rosetta cells harboring the plasmid were grown and induced for protein expression as described earlier (42). RAD51AP1-N88 was purified by tandem purification using Ni-NTA resin (ThermoScientific) first, as described (42).…”

“…For purification of the RAD51AP1-N88 protein, E. coli Rosetta cells harboring the plasmid were grown and induced for protein expression as described earlier (42). RAD51AP1-N88 was purified by tandem purification using Ni-NTA resin (ThermoScientific) first, as described (42). Eluted protein was incubated with pre-equilibrated Strep-Tactin Superflow Plus (Qiagen) and gentle rotation at 4°C for 1 h in binding buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 2 mM DTT.…”