Background:The single-strand DNA-binding protein, replication protein A (RPA), is essential for replication, repair, and recombination. Results: This study elucidates the molecular defects of RPA mutants that cause cell cycle arrest. Conclusion: These mutants are active in DNA replication but not in DNA repair because of altered binding to short ssDNA intermediates. Significance: This study indicates that DNA repair and replication require different RPA-DNA interactions.