Banerjee et al. 10.1073/pnas.0504063102. |
Supporting Figure 5
Supporting Table 1
Supporting Figure 6
Supporting Table 2
Supporting Table 3
Supporting Table 4
Supporting Figure 5
Fig. 5.
The overexpression of yKu70-yKu80 does not alter capability of DNA repair. (A) The level of overexpression of yKu70-yKu80 was determined by Western blotting with antibodies against yeast Ku70 and Ku80. (B) The overexpression of yKu70-yKu80 in yeast does not change the viability in response to methyl methane sulfonate (MMS). Exponentially growing cells were exposed to the indicated dose of MMS for 2 h, and the surviving percentage was calculated by comparing surviving colonies after treatment with colonies formed without MMS treatment. (C) The Ku overexpression does not affect the nonhomologous end joining (NHEJ) activity. The Ku overexpression in the rad52 strain showed similar MMS sensitivity with the rad52 strain carrying control plasmids, whereas the rad52 lig4 strain carrying control plasmids showed greater sensitivity to MMS. Three different strains were plated on synthetic dextrose plate with all amino acids except tryptophane and histidine and the same plate carrying 0.005% MMS. Then, the surviving percentage was calculated by comparing the number of colonies from both plates.Fig. 6.
The overexpression of yKu70-yKu80 in wild type did not change the telomere size. Ku overexpression in the pif1-m2 strain slightly reduced the telomere size in several clones, which had escaped the S phase growth arrest. Control and yKu70/80 indicate strains carrying either pYES3CT and pRS425 plasmids or pKJM5 (yKU80) and pKJM16 (yKU70), respectively.Table 2. Growth defects caused by the overexpression of yKu70-yKu80 heterodimer in the pif1-m2 strain were not rescued by mutations in the yeast caspase gene yca1 or the nonhomologous end-joining ligase gene lig4
Relevant genotype | yKu70-yKu80 plasmids | Growth condition | |
Glucose | Galactose | ||
pif1-m2 pif1-m2 | - + | +++++++ +++++ | ++++++ + |
pif1-m2 yca1 pif1-m2 yca1 | - + | +++++++ +++++ | ++++++ + |
pif1-m2 lig4 pif1-m2 lig4 | - + | ++++++ ++++++ | ++++++ + |
pif1-m2 rad24 pif1-m2 rad24 | - + | +++++++ ++++++ | ++++++ +++++ |
+ or – in the yKu70-yKu80 plasmids column represent the presence or absence of yKu70 and yKu80 expression, respectively. The + symbols in growth condition columns represent the level of growth under the condition indicated.
Table 3. The slow-growth phenotype by the overexpression of the yKu70-yKu80 heterodimer is rescued by mutations causing defects in Okazaki fragment maturation
+ or – in the yKu70-yKu80 plasmids column represent the presence or absence of yKu70 and yKu80 expression, respectively. The + symbols in the growth condition columns represent the level of growth under the condition indicated.
Table 4. Saccharomyces cerevisiae strains used in this study
Strains | Relevant genotype | Plasmid number | Source |
YKJM1220 | pif1-m2 | pYES3CT, pRS425 | This study |
YKJM1222 | Wild type | This study | |
YKJM1225 | Wild type | pKJM5, pKJM16 | This study |
YKJM1227 | pif1-m2 | This study | |
YKJM1264 | Wild type | pYES3CT | This study |
YKJM1407 | mec1::HIS3, sml1::KAN | pYES3CT, pRS425 | This study |
YKJM1409 | mec1::HIS3, sml1::KAN | pKJM5, pKJM16 | This study |
YKJM1411 | rad52::HIS3 | pYES3CT, pRS425 | This study |
YKJM1413 | rad52::HIS3 | pKJM5, pKJM16 | This study |
YKJM1415 | rfa1-t33 | pYES3CT, pRS425 | This study |
YKJM1417 | rfa1-t33 | pKJM5, pKJM16 | This study |
YKJM1419 | mre11::HIS3 | pYES3CT, pRS425 | This study |
YKJM1421 | mre11::HIS3 | pKJM5, pKJM16 | This study |
YKJM1482 | rad27::KAN | This study | |
YKJM1484 | rad27::KAN | pKJM5, pKJM16 | This study |
YKJM1488 | pif1-m2, rad24::HIS3 | pYES3CT, pRS425 | This study |
YKJM1490 | pif1-m2, rad24::HIS3 | pKJM5, pKJM16 | This study |
YKJM1651 | pif1-m2, yca1::HIS3 | pYES3CT, pRS425 | This study |
YKJM1653 | pif1-m2, yca1::HIS3 | pKJM5, pKJM16 | This study |
YKJM2004 | rad24::HIS3 | pYES3CT, pRS425 | This study |
YKJM2006 | rad24::HIS3 | pKJM5, pKJM16 | This study |
YKJM2095 | sgs1::HIS3 | pYES3CT, pRS425 | This study |
YKJM2097 | sgs1::HIS3 | This study | |
YKJM2098 | rad9::HIS3 | pYES3CT, pRS425 | This study |
YKJM2100 | rad9::HIS3 | pKJM5, pKJM16 | This study |
YKJM2103 | lig4::HIS3 | pYES3CT, pRS425 | This study |
YKJM2105 | lig4::HIS3 | This study | |
YKJM2106 | mad2::HIS3 | pYES3CT, pRS425 | This study |
YKJM2108 | mad2::HIS3 | This study | |
YKJM2151 | dpb11-1 | pYES3CT, pRS425 | This study |
YKJM2153 | dpb11-1 | This study | |
YKJM2163 | pif1-m2, lig4::HIS3 | pYES3CT, pRS425 | This study |
YKJM2164 | pif1-m2, lig4::HIS3 | pKJM5, pKJM16 | This study |
YKJM2416 | Wild type | pKJM375 | This study |
YKJM2602 | elg1::HIS3 | pYES3CT, pRS425 | This study |
YKJM2604 | elg1::HIS3 | pKJM5, pKJM16 | This study |
YKJM2719 | pol3-01 | pYES3CT, pRS425 | This study |
YKJM2720 | pol3-01 | pKJM5, pKJM16 | This study |
HO Strain | |||
YKJM1231 | Wild type | pYES3CT, pRS425, pRS313 | This study |
YKJM1233 | Wild type | pYES3CT, pRS425, pKJM20 | This study |
YKJM1235 | Wild type | pKJM5, pKJM16, pRS313 | This study |
YKJM1237 | Wild type | pKJM5, pKJM16, pKJM20 | This study |
All strains are isogenic to RDKY3615 [ura3-52, leu2D1, trp1D63, his3D200, lys2DBlg, hom3-10, ade2D1, ade8, YEL069::URA3] or YKJM941 [ura3::KAN, leu2D1, trp1D63, his3D200, lys2DBlg, hom3-10, ade2D1, ade8, HO::hisG, sit::URA3-HO] for general assays or an HO-inducible assay, respectively, except mutations and plasmids described. pKJM5 (LEU2), pKJM6 (URA3), pKJM16 (TRP1), pKJM375 (TRP1), and pKJM376 (TRP1) encode yKU80, yKU70, or MCM1 under a galactose promoter, respectively. pKJM20 (HIS3) encodes an HO endonuclease under a galactose-inducible promoter.