The available SRL3 deletion strain of Saccharomyces cerevisiae contains a truncation of DNA damage tolerance protein Mms2: Implications for Srl3 and Mms2 functions

Abstract

A screen of the commercially available collection of haploid deletion mutants of Saccharomyces cerevisiae for spontaneous mutator mutants newly identified a deletion of SRL3. This gene had been previously isolated as a suppressor of lethality of checkpoint kinase deletions if overexpressed. We found DNA damage sensitivity and extended checkpoint arrests to be associated with this strain. However, when crossed to wild-type, a mutant gene conferring these phenotypes was found to segregate from the SRL3 deletion. The mutation was identified as a C-terminal truncation of Mms2, an E2 ubiquitin conjugating enzyme involved in error-free replicative bypass of lesions. This confirmed an earlier report that Mms2 may be required to restrain error-prone polymerase ζ activity and underscored that residues of the C-terminus are necessary for Mms2 function. Srl3, on the other hand, does not appear to influence DNA damage sensitivity or spontaneous mutability if deleted. However, the absence of these phenotypes does not contradict its likely role as a positive regulator of dNTP levels.

Keywords: Checkpoints; DNA Damage Tolerance; DNA Repair; Mutagenesis; Yeast; dNTP Pools.

Figure 1

Survival of BY4741 srl3Δ uvs following exposure to UV radiation (A), MMS (B) and streptonigrin (C). Additionally, the UV survival of strains BY4741 rnr1Δ, rnr3Δ and rnr4Δ are shown (A). Logarithmic-phase cells were treated, as described in Material and Methods, and the fraction of colony forming cells was plotted as a function of dose.

Figure 2

Checkpoint arrest duration in srl3Δ uvs in comparison to wild type. Exit from G1 (A), S (B) and M phase (C) following UV radiation treatment (80 J/m²) was analyzed by determining the fraction of small budded cells in liquid medium (A) or by the emergence of microcolonies (with more than two cells) on plates (B, C).

Figure 3

UV sensitive phenotype of srl3Δ uvs is caused by a mutation in MMS2 (= uvs). A. Segregation of UV sensitivity. Following crossing of srl3Δuvs to wild-type, spores were isolated containing SRL3 deletion as indicated by the transplacement marker kanMX4, conferring G418 resistance. As shown, SRL3 deletion and UV sensitivity did not cosegregate. B, C. Epistasis analysis with uvs. The unknown mutation (uvs) was combined with deletions of RAD5 and RAD50. In logarithmic-phase cells, UV (B) and MMS (C) sensitivities of double mutants were determined and compared to single mutant sensitivities. D. A uvs strain was mated with strains carrying deletions of RAD50, MRE11, XRS2, RAD5, UBC13 or MMS2. UV and MMS sensitivities of the diploid strains were characterized in gradient assays. Enhanced sensitivity was only found for the mms2Δ uvs combination.

Figure 4

MMS2 DNA and protein sequence. The mutation identified in srl3Δ uvs is indicated.