RAD51AP1-deficiency in vertebrate cells impairs DNA replication

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• RAD51AP1 is crucial for homologous recombination (HR) by supporting RAD51 and DMC1 recombinases.
• RAD51AP1 knockdown in human cells increases DNA damage sensitivity but doesn't affect RAD51 foci formation.

Purpose of the Study:

• To investigate the phenotype of complete RAD51AP1 inactivation in vertebrate cells using a chicken DT40 model.
• To elucidate the role of RAD51AP1 in DNA repair, replication stress, and cellular viability.

Main Methods:

• Generated RAD51AP1-deficient DT40 cell lines.
• Assessed cytotoxicity after exposure to cisplatin and ionizing radiation.
• Performed complementation assays with GgRAD51AP1 and HsRAD51AP1.
• Analyzed RAD51 foci dynamics and replication fork progression in human and chicken cells.

Main Results:

• RAD51AP1 inactivation did not affect cell viability or proliferation in undamaged cells but increased sensitivity to DNA damaging agents.
• RAD51AP1 deficiency significantly slowed the resolution of DNA damage-induced RAD51 foci.
• RAD51AP1 is essential for maintaining replication fork speed and counteracting replication stress.
• RAD51AP1-deficient cells exhibited slower replication fork elongation but increased origin firing.

Conclusions:

• Complete RAD51AP1 inactivation impairs DNA repair and replication fork stability, leading to increased sensitivity to genotoxic stress.
• RAD51AP1 plays a vital role in managing replication stress, which may explain its overexpression in tumors.
• Targeting RAD51AP1 function presents a potential strategy for cancer therapy.