NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

Abstract

NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.

Role of RAD51AP1 in homologous recombination DNA repair and carcinogenesis

“…NUCKS1, however, does not harbor any sequence that resembles the RAD51AP1 C-terminal RAD51 interaction domain. Consistent with this observation, NUCKS1 and RAD51 do not physically interact [24]. …”

“…However, RAD51AP1 has the highest affinity for branched DNA substrates and the D-loop, indicative of a role in the formation of DNA intermediates in the HR reaction [15, 16, 20, 22, 24, 25]. Via its DNA binding attribute, RAD51AP1 stimulates the RAD51-mediated D-loop reaction in amounts sub-stoichiometric to that of RAD51 [15, 16].…”

“…RAD51AP1 shares extensive (28% identical and 11% similar) sequence homology with the NUCKS1 (Nuclear Casein Kinase and Cyclin-dependent Kinase Substrate 1) protein, indicating that these proteins are paralogs [24]. NUCKS1, however, does not harbor any sequence that resembles the RAD51AP1 C-terminal RAD51 interaction domain.…”

“…As demonstrated in DNA combing experiments, loss of RAD51AP1 leads to slowing of DNA replication fork progression and elevated DNA replication stress [20, 24]. To resolve these replication abnormalities, RAD51AP1 -deficient cells increase their usage of replication origins whereby the timely completion of DNA replication is ensured and further replication stress is minimized [20, 24].…”

“…To resolve these replication abnormalities, RAD51AP1 -deficient cells increase their usage of replication origins whereby the timely completion of DNA replication is ensured and further replication stress is minimized [20, 24]. These results show that RAD51AP1 is required for replication fork stability and the avoidance of replication stress.…”