of proteins on nascent DNA; OZF, only-zinc finger; PFA, paraformaldehyde; PI, propidium iodide; RT, room temperature; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; WB, western blot. Abstract DNA replication is essential for cell proliferation and is one of the cell cycle stageswhere DNA is more vulnerable. Replication stress is a prominent property of tumor cells and an emerging target for cancer therapy. Although it is not directly involved in nucleotide incorporation, Claspin is a protein with relevant functions in DNA replication. It harbors a DNA-binding domain that interacts preferentially with branched or forked DNA molecules. It also acts as a platform for the interaction of proteins related to DNA damage checkpoint activation, DNA repair, DNA replication origin firing, and fork progression. In order to find new proteins potentially involved in the regulation of DNA replication, we performed a two-hybrid screen to discover new Claspin-binding proteins. This system allowed us to identify the zinc-finger protein OZF (ZNF146) as a new Claspin-interacting protein. OZF is also present at replication forks and co-immunoprecipitates not only with Claspin but also with other 6908 | FEU Et al.

Solving the problems that replication forks encounter when synthesizing DNA is essential to prevent genomic instability. Besides their role in DNA repair in the G2 phase, several homologous recombination proteins, specifically RAD51, have prominent roles in the S phase. Using different cellular models, RAD51 has been shown not only to be present at ongoing and arrested replication forks but also to be involved in nascent DNA protection and replication fork restart. Through pharmacological inhibition, here we study the specific role of RAD51 in the S phase. RAD51 inhibition in non-transformed cell lines did not have a significant effect on replication fork progression under non-perturbed conditions, but when the same cells were subjected to replication stress, RAD51 became necessary to maintain replication fork progression. Notably, the inhibition or depletion of RAD51 did not compromise fork integrity when subjected to hydroxyurea treatment. RAD51 inhibition also did not decrease the ability to restart, but rather compromised fork progression during reinitiation. In agreement with the presence of basal replication stress in human colorectal cancer cells, RAD51 inhibition reduced replication fork speed in these cells and increased γH2Ax foci under control conditions. These alterations could have resulted from the reduced association of DNA polymerase α to chromatin, as observed when inhibiting RAD51. It may be possible to exploit the differential dependence of non-transformed cells versus colorectal cancer cells on RAD51 activity under basal conditions to design new therapies that specifically target cancer cells.

Solving the problems that replication forks encounter when synthesizing DNA is essential to prevent genomic instability. Besides their role in DNA repair in the G2 phase, several homologous recombination proteins, specifically Rad51, have prominent roles in the S phase. Using different cellular models, Rad51 has been shown not only to be present at ongoing and arrested replication forks but also to be involved in nascent DNA protection and replication fork restart. Through pharmacological inhibition, here we study the specific role of Rad51 in the S phase. Rad51 inhibition in non-transformed cell lines did not have a major effect on replication fork progression under non-perturbed conditions, but when the same cells were subjected to replication stress, Rad51 became necessary to maintain replication fork progression. Notably, the inhibition or depletion of Rad51 did not compromise fork integrity when subjected to hydroxyurea treatment. Rad51 inhibition also did not decrease the ability to restart, but rather compromised, fork progression during reinitiation. In agreement with the presence of basal replication stress in human colorectal cancer cells, Rad51 inhibition reduced replication fork speed in these cells and increased γH2Ax foci under control conditions. These alterations could have resulted from the reduced association of DNA polymerase α to chromatin, as observed when inhibiting Rad51. It may be possible to exploit the differential dependence of non-transformed cells versus colorectal cancer cells on Rad51 activity under basal conditions to design new therapies that specifically target cancer cells.