Analysis of Yeast Genes Influencing the Lethality of DNA Damage Related Checkpoint Mutants

The purpose of this study was to determine the functions of Hug1 and Srl3. It has been reported that HUG1 or SRL3 deletion rescues the lethality of a DNA damage checkpoint gene deleted mutant, mec1Δ. It is known that the lethality of mec1Δ can be rescued by high dNTP levels. To elucidate the functions of these proteins, the phenotypes of hug1Δ and srl3Δuvs, as well as the transcript profile of hug1Δ were analyzed. Novel phenotypes of hug1Δ were uncovered: resistance to oxidative stress or heat shock, earlier arrest in G1/G0 phase, defect in hydroxyurea-induced filamentation, and slow growth inresponse to combined stresses of hydroxyurea and reduced dextrose content. These phenotypes correlate with a transcription profile that indicated altered stress responses in hug1Δ as compared to WT. We assumed that the reason for many constitutively expressed stress-related transcripts is a higher dNTP level in hug1Δ compared to WT. The similarities in the phenotypes of dif1Δ and sml1Δ to those of hug1Δ support the assumption. The phenotypes of dif1Δ and sml1Δ were studied since Dif1 and Sml1 are known inhibitors of ribonucleotide reductase activity. Furthermore, Dif1, Sml1, and Hug1 are considered proteins that evolved from the same ancestor protein. Initially, Srl3 was a protein of special interest because the commercially available srl3Δ mutant causes high spontaneous mutation rates and sensitivity to UV light. However, during the course of this study, it was found that the two phenotypes originated from a second, unrelated mutation in srl3Δ strain. Through complementation test and sequencing, this mutation was identified as a nonsense mutation of MMS2, a gene involved in post-replication repair.