Abstract: The cytolethal distending toxins (CDTs) are secreted virulence proteins produced by several bacterial pathogens and the subunit CdtB has the ability to create DNA lesion, primarily DNA single-stranded breaks (SSBs) in vitro, and cause cell cycle arrest, cellular distension and cell death in both mammalian and yeast cells. To elucidate the components of the mechanisms underlying the response to CdtB-induced DNA lesions, a CdtB expression plasmid was transformed into a series of diploid yeast strains harboring deletions in 4708 nonessential genes. 4706 of these clones were successfully transformed, which we have now designated as a systematic transformation array (STA), and were subsequently screened. We identified 61 sensitive strains from the STA whose deleted genes can be categorized into a number of groups including DNA metabolism, chromosome segregation, vesicular traffic, RNA catabolism, protein translation, morphogenesis, nuclear transport, and one unknown ORF. However, only 28 of these strains were found to be sensitive to HO endonuclease, which is known to create DNA double-stranded break (DSB), suggesting that CdtB-induced DNA lesion is not similar to the direct DSB. Amazingly, CdtB expression elicits severe growth defects in haploid yeast cells, but only marginal defects in diploid yeast cells. The presence and absence of genes known to be involved in DNA repair in these genome-wide data reveal that CdtB-induced DNA damage is specifically repaired well in diploid by homologous recombination but not by other repair mechanisms. Our current results provide insights into how CdtB pathogenesis is linked to eukaryotic cellular functions.