The antitumor agent camptothecin targets DNA topoisomerase I by reversibly stabilizing a covalent enzyme-DNA intermediate. The subsequent collision of DNA replication forks with these drug-enzyme-DNA complexes produces the cytotoxic DNA lesions that signal cell cycle arrest and ultimately lead to cell death. Despite intense investigation, the character of the lesions produced and the repair processes that resolve the damage remain poorly defined. A yeast genetic screen was implemented to isolate conditional mutants with enhanced sensitivity to DNA topoisomerase I-mediated DNA damage. Cells exhibiting temperature-sensitive growth in the presence of the DNA topoisomerase 1 mutant, Top1T722Ap, were selected. Substitution of Ala for Thr722 increases the stability of the covalent Top1T722Ap-DNA intermediate, mimicking the cytotoxic action of camptothecin. Two mutants isolated, cdc45-10 and dpb11-10, exhibited specific defects in DNA replication and a synthetic lethal phenotype in the absence of DNA damaging agents. The accumulation of Okazaki fragments under nonpermissive conditions suggests a common function in promoting processive DNA replication through polymerase switching. These results provide a mechanistic basis for understanding the cellular processes involved in the resolution of DNA damage induced by camptothecin and DNA topoisomerase I.