The available SRL3 deletion strain of Saccharomyces cerevisiae contains a truncation of DNA damage tolerance protein Mms2: Implications for Srl3 and Mms2 functions
E Kim, W Siede
checkpoints, dna damage tolerance, dna repair, dntp pools, mutagenesis, yeast
E Kim, W Siede. The available SRL3 deletion strain of Saccharomyces cerevisiae contains a truncation of DNA damage tolerance protein Mms2: Implications for Srl3 and Mms2 functions. The Internet Journal of Microbiology. 2009 Volume 8 Number 1.
A screen of the commercially available collection of haploid deletion mutants of
A largely conserved network of proteins is in place to prevent lethal and mutagenic consequences of DNA damage and replication stress in eukaryotes (Friedberg et al., 2006). In this context, the regulation of dNTP levels has attracted considerable attention and many details surrounding the regulation of the heterotetrameric ribonucleotide reductase (RNR) are known in
In budding yeast, an elevated dNTP level can prevent the lethality of checkpoint kinase (Mec1 or Rad53) deletions. These checkpoint kinases are necessary for cell cycle arrest and facilitation of DNA repair (Friedberg et al., 2006; Nyberg et al., 2002). Elevated dNTP levels can be achieved by deletion of negative regulators such as Sml1, Ctr1 or Dif1 or by overexpression of RNR subunit Rnr1 (Desany et al., 1998; Lee et al., 2008; Zhao et al., 1998).
Additional genes of unknown function have been identified through a selection for genes that, if overexpressed, suppress the lethality of a
Materials and Methods
Yeast strains and strain construction
Most haploid strains were derived from BY4741 (
Determination of spontaneous mutation rates
Parallel cultures were inoculated with 2,000 cells in 4 ml YPD liquid medium (1% yeast extract, 2% peptone, 2% dextrose). Cultures were incubated at 30°C (75 rpm) until the titer of each culture reached at least 1 × 108 cells/ml. From each culture, 2 x 107 cells were plated on canavanine plates (containing 1.4% agar, 2% dextrose, 6.7 g/l yeast nitrogen base w/o amino acid and w/o ammonium sulfate, complete supplement mixture minus arginine [CSM-Arg; Sunrise Science] and 60 mg/l canavanine sulfate) to determine the frequency of canavanine-resistant mutants. Titer of all colony-forming cells was determined on medium of the same composition without canavanine. Plates were incubated at 30°C until formation of colonies (three to four days). The median mutant colony number was used to determine relative spontaneous mutation frequencies according to the method of Lea and Coulson, as described (Lea and Coulson, 1949; von Borstel, 1978).
For UV irradiation, appropriate dilutions of cell suspensions were plated on YPD plates and irradiated with 254 nm UV (germicidal lamp). For methyl methane sulfonate (MMS) and streptonigrin (both from Sigma Chemicals), liquid cultures were treated for 30 minutes at 30°C with various concentrations of the chemicals. Cells were washed, then appropriate dilutions of untreated and treated cells were plated on YPD plates. Plates were incubated at 30°C until surviving cells formed colonies (about 3 to 4 days). Colonies on each plate were counted and the surviving fractions were calculated. Surviving fraction is defined as titer of colony forming cells with treatment divided by the titer of colony forming cells without treatment.
Checkpoint assays following UV irradiation
To measure exit from G1 phase arrest, logarithmic-phase phase cultures of a titer of approximately 1 × 107 cells/ml were first synchronized in G1 by exposure to alpha-factor (1 mg/ml stock solution, US Biological) at 5 µg/ml in fresh YPD. After cells were incubated at 30°C for 1 hour and 15 minutes, another aliquot of alpha-factor was added up to a final concentration of 10 µg/ml and cells were incubated for another 30-45 minutes. After microscopic confirmation of G1 arrest, 8 x 107 cells were spun down, washed, briefly sonicated and resuspended with 8 ml of sterile water. 4 ml of this cell suspension was transferred to a sterile petridish (6 cm diameter) and irradiated with 254 nm UV (80 J/m2) under constant stirring. 4 ml of irradiated and 4 ml of non-irradiated cells were centrifuged and each sample was resuspended with 4 ml of YPD. During incubation at 30°C, the percentage of small-budded cells was microscopically determined every 10 to 15 minutes.
To measure exit from S phase arrest
To measure exit from M phase arrest, logarithmic-phase cultures were synchronized with nocodazole (at 10 µg/ml, US Biological) for 3 hours at 30°C, washed, briefly sonicated, streaked on YPD and observed as described above. Since the microscopic determination of small buds is difficult on solid media, this determination of UV-induced S and M phase arrest may also include the arrest in the subsequent G1 phase.
The commercially available collection of deletion mutants of non-essential genes of a haploid yeast strain (BY4741) was screened for strains with higher spontaneous mutation frequencies, using canavanine resistance as a forward mutation marker (Gong and Siede, 2009). While this screen was similar to the one described earlier by others (Huang et al., 2003), two novel genes were identified:
We focused on
Data shown are from representative single experiments, except for strain
Also, determination of UV sensitivity in
UV sensitivity was not due to a checkpoint defect. When cells were synchronized in G1 with alpha-factor, in S with HU and in M with nocodazole before treatment with UV, a more extended arrest was found in all tested cell cycle stages (Fig. 2 A, B, C).
Prompted by the fact that the second commercially available
In order to identify possible candidate genes whose mutation may result in the observed phenotypes, we performed epistasis analyses with well-characterized single gene deletions conferring similar phenotypes. Briefly, in epistasis analysis, an uncharacterized mutant gene affecting DNA damage sensitivity is assigned to the same DNA repair or tolerance pathway as a characterized mutant gene if the double mutant’s phenotype is not more severe than that of the most sensitive single mutant (Brendel and Haynes, 1973). Given the sensitivity of the
Next, we identified candidate genes of the error-free subpathway of the Rad6 epistasis group or the MRN complex whose mutation may account for the
In a screen of the commercially available deletion collection in the haploid yeast strain BY4741, deletions of
Further analysis, however, revealed that the described phenotype in this particular strain originated from another unlinked mutation that results in truncation of the damage tolerance protein Mms2. An
Mms2 functions in a sub-pathway of the DNA-damage tolerance pathway (Rad6 group) that facilitates replication of a damaged template (Andersen et al., 2008). Mms2 is a ubiquitin-conjugating (E2) variant enzyme that forms a heterodimer with the E2 enzyme Ubc13 (VanDemark et al., 2001) and interacts with the ubiquitin-ligase Rad5 (Broomfield et al., 1998; Gangavarapu et al., 2006; Hofmann and Pickart, 1999; Torres-Ramos et al., 2002; Xiao et al., 1998). Their primary target during lesion bypass appears to be PCNA that is subject to polyubiquitination (Hoege et al., 2002). Error-free template switching is postulated as the used mechanism of lesion bypass (Branzei et al., 2008). There is one earlier report of higher spontaneous mutation rates in
Given the role of Mms2 in the replicative bypass of UV lesion, extended checkpoint arrest of its mutant in S-phase can easily be rationalized. This is not the case with the observed extension of G1 and M phase arrest where the assumed bypass mechanism cannot easily contribute to UV resistance. We hypothesize that there may be an unknown role of Rad5-Mms2-Ubc13 during nucleotide excision repair. The observed extension of checkpoint arrest was independent of
We have no indication for any functional connection between Srl3 and Mms2 and assume that the appearance of both mutations together is a coincidence. For example, continued propagation of