The human HLTF gene is located on chromosome 3q24 and is frequently amplified in squamous cell lung carcinomas. HLTF is the human ortholog of the Saccharomyces cerevisiae RAD5 gene, a DNA helicase and ubiquitin ligase that functions in post replication DNA damage repair. Overexpression of RAD5 results in slow growth and genome instability. We used Selective Ploidy Ablation (SPA), a rapid high throughput plasmid screening protocol, to identify Saccharomyces cerevisiae genes whose function becomes essential when RAD5 is overexpressed. Many DNA replication and DNA double strand break repair genes were identified covering multiple steps in these processes, including replication fork protection, lagging strand DNA synthesis, DNA end resection, homology dependent strand invasion and resolution of Holliday junction intermediates. Curiously, genes regulating the process of post replication repair (PRR) are unaffected in the screen. Cells encountering DNA damage that results in replication fork stalling enter the PRR pathways via modification of PCNA/Pol30 by ubiquitylation. Using multiple mutant backgrounds, we confirmed that ubiquitylation of PCNA is not necessary for the growth suppression resulting from RAD5 overexpression. Since Rad5 is a multidomain polypeptide containing helicase, ubiquitin ligase and DNA binding functions, we investigated the contribution of these domains to RAD5 toxicity. Mutant protein constructs were screened using the high throughput SPA method to transfer each RAD5 mutant into an array of sensitized mutant strains. Automated quantitative analysis of the growth curves was used to define the effects of individual RAD5 mutants in strains deficient for specific repair functions. We find that the individual domains of overexpressed RAD5 have separate contributions to cell toxicity in different genetic backgrounds. For example, we have uncovered instances where deletion of a specific Rad5 domain increases toxicity, suggesting that it normally performs a protective function. As the function of Rad5 is conserved in human cells, the identified SDL interactions may help to develop targeted therapeutic approaches for squamous cell carcinomas of the lung.