A genome-wide screen for methyl methanesulfonate-sensitive mutants reveals genes required for S phase progression in the presence of DNA damage

Abstract

We performed a systematic screen of the set of approximately 5,000 viable Saccharomyces cerevisiae haploid gene deletion mutants and have identified 103 genes whose deletion causes sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS). In total, 40 previously uncharacterized alkylation damage response genes were identified. Comparison with the set of genes known to be transcriptionally induced in response to MMS revealed surprisingly little overlap with those required for MMS resistance, indicating that transcriptional regulation plays little, if any, role in the response to MMS damage. Clustering of the MMS response genes on the basis of their cross-sensitivities to hydroxyurea, UV radiation, and ionizing radiation revealed a DNA damage core of genes required for responses to a broad range of DNA-damaging agents. Of particular significance, we identified a subset of genes that show a specific MMS response, displaying defects in S phase progression only in the presence of MMS. These genes may promote replication fork stability or processivity during encounters between replication forks and DNA damage.

Fig 1.

High-throughput MMS screen. The complete set of haploid yeast deletion mutants was arrayed in duplicate onto 16 plates and pinned onto YPD media or YPD + 0.035% MMS (array plate 11 of 16 is shown). Putative MMS-sensitive mutants lead to the formation of smaller colonies when grown on MMS-containing media.

Fig 2.

Confirmation of MMS sensitivity. Putative MMS-sensitive strains were grown in YPD overnight at 30°C. Serial 10-fold dilutions were spotted onto YPD, YPD + 0.035% MMS, or YPD + 200 mM HU and incubated at 30°C for 3 days. Strains in bold were scored as sensitive. A rad53 mutant was used as a positive control.

Fig 3.

S phase progression analysis of selected MMS mutants. Cells were arrested in G₁ and released in either the presence or absence of 0.035% MMS. Shaded histograms represent the cell cycle distribution of asynchronous culture before cell cycle synchronization. Overlaid histograms represent the cell cycle distribution after release from G₁ arrest ± 0.035% MMS for the indicated times.