Budding Yeast Hed1 Down Regulates the Mitotic Recombination Machinery When Meiotic

Budding Yeast Hed1 Down Regulates the Mitotic Recombination Machinery when Meiotic Recombination Is Impaired

Hideo Tsubouchi and G. Shirleen Roeder

Supplemental Research Data

Yeast strains

Genotypes of yeast strains used are listed in Table S1. The entire RAD51 ORF was replaced with clonNAT(Goldstein and McCusker 1999) to generate rad51::clonNAT by PCR-mediated gene disruption. red1::URA3, dmc1::KAN, dmc1::URA3, dmc1::LEU2, rad51::URA3 and hop2::ADE2 were described previously (Bishop et al. 1992; Shinohara et al. 1992; Rockmill et al. 1995; Smith and Roeder 1997; Tsubouchi and Roeder 2002; Tsubouchi and Roeder 2004).

The strains used in each Figure are the following. In Figure 1A, TBR1224 carrying YCplac22-red1-22 and YEp24-RED1, YEp24 or YEplac195-HED1 were used (Botstein et al. 1979; Gietz and Sugino 1988). In Figure 1C, TBR324 carrying YCplac22-red1-22 and TBR1306 carrying YCplac22-red1-22 were used. In Figure 2A, S3246, TBR249, TBR310, TBR869, TBR1086, TBR1419, TBR1543 and TBR1587 were used. In Figure 2B, NHY290, TBR2111, TBR2112, TBR2113, TBR2114, TBR2090, TBR2091 and TBR2092 were used. In Figure 2C, TBR869 and TBR1587 carrying YEp24, YEp24-RAD51, or YEpFAT4-RAD51(Tsubouchi and Roeder 2003) were used. In Figure 3, TBR249, TBR310, TBR1419, TBR1506, TBR1507 and TBR1543 were used. In Figure 4B, NHY290, TBR2090, TBR2091 and TBR2092 were used. In Figure 4C, TBR2111, TBR2112, TBR2113 and TBR2114 were used. In Figure 5, strains used were TBR1567 (A and B), TBR1608 (C), TBR1520 (D) and PJ69-4A (E). In Figure 6A and 6B, HTY223 and TBR1664 were used. In Figure 6D, TBR1701 and TBR1703 were used.

Supplementary Table 1. Yeast strains
Strain / Genotype
TBR1224 / MATa leu2 his4 ade2 THR1trp1 can1 ura3 cyh2 red1::KAN
MAT leu2 his4 ade2 thr1 trp1 CAN1 ura3 CYH2 red1::KAN
S3246 / MATa leu2-3,112 his4-260 thr1-4 ura3-1 trp1-289 ade2-1
MAT leu2-3,112 his4-260 thr1-4 ura3-1 trp1-289 ade2-1
TBR249 / S3246 but his4-260/his4-Hpa andhomozygous dmc1::URA3
TBR310 / S3246 but homozygous hop2::ADE2
TBR324 / S3246 but homozygous red1::URA3
TBR869 / S3246 but homozygous dmc1::KAN
TBR1086 / S3246 but homozygous hed1::KAN
TBR1306 / S3246 but homozygous red1::URA3 hed1::KAN
TBR1419 / S3246 but homozygous hop2::ADE2 hed1::KAN
TBR1506 / S3246 but homozygous dmc1::URA3 rad51::URA3
TBR1507 / S3246 but homozygous dmc1::URA3 rad51::URA3 hed1::KAN
TBR1520 / S3246 but homozygous hop2::ADE2 HED1-GFP::KAN
TBR1543 / S3246 but homozygous dmc1::URA3 hed1::KAN
TBR1567 / S3246 but homozygous HED1-GFP::KAN
TBR1587 / S3246 but homozygous dmc1::KAN hed1::KAN
TBR1608 / S3246 but homozygous rad51::URA3 HED1-GFP::KAN
NHY290 / MATa leu2::hisG HIS4::LEU2-(NBam) ho::hisG ura3(∆Pst-Sma)
MAT leu2::hisG his4X::LEU2(NBam)-URA3 ho::hisG ura3(∆Pst-Sma)
TBR2090 / NHY290 but homozygous dmc1::KAN
TBR2091 / NHY290 but homozygous hed1::HYG
TBR2092 / NHY290 but homozygous dmc1::KANhed1::HYG
TBR2111 / NHY290 but homozygousrad51::clonNAT
TBR2112 / NHY290 but homozygous rad51::clonNAT dmc1::KAN
TBR2113 / NHY290 but homozygous rad51::clonNAT dmc1::KANhed1::HYG
TBR2114 / NHY290 but homozygous rad51::clonNAT hed1::HYG
HTY223 / MATura3-52 leu2-3,112 trp1-289 ade5-1 his7-2
TBR1664 / HTY223but KAN-GAL1-HED1
TBR1701 / MATa-incho∆ hml::ADE1 hmr::ADE1 ade1 leu2-3, 112 lys5 trp1::hisG ura3-52 ade3::GAL10::HO with pJH273
TBR1703 / TBR1701 but KAN-GAL1-HED1
PJ69-4A / MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4∆ gal8∆ LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ

Both parents of S3246(Tsubouchi and Roeder 2003) are isogenic with BR1919-8B (Rockmill and Roeder 1990). TBR1224 is congenic with S3246. NHY290 (Hunter and Kleckner 2001) and its derivatives are SK1 strains. HTY223 was described previously (Tsubouchi and Ogawa 1998). TBR1701 is isogenic with JKM186 (Lee et al. 1998). PJ69-4A was described previously (James et al. 1996). Isogenic strains were derived by transformation and/or genetic crosses.

Supplementary References

Bishop, D.K., Park, D., Xu, L., and Kleckner, N. 1992. DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell69: 439-456.

Botstein, D., Falco, S.C., Stewart, S.E., Brennan, M., Scherer, S., Stinchcomb, D.T., Struhl, K., and Davis, R.W. 1979. Sterile host yeast (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene8: 17-24.

Gietz, R.D. and Sugino, A. 1988. New yeast - Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene74: 527-534.

Goldstein, A.L. and McCusker, J.H. 1999. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast15: 1541-1553.

Hunter, N. and Kleckner, N. 2001. The single-end invasion: an asymmetric intermediate at the double-strand break to double-Holliday junction transition of meiotic recombination. Cell106: 59-70.

James, P., Halladay, J., and Craig, E.A. 1996. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics144: 1425-1436.

Lee, S.E., Moore, J.K., Holmes, A., Umezo, K., Kolodner, R.D., and Haber, J.E. 1998. Saccharomyces Ku70, Mre11/Rad50, and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell94: 399-409.

Rockmill, B. and Roeder, G.S. 1990. Meiosis in asynaptic yeast. Genetics126: 563-574.

Rockmill, B., Sym, M., Scherthan, H., and Roeder, G.S. 1995. Roles for two RecA homologs in promoting meiotic chromosome synapsis. Genes Dev.9: 2684-2695.

Shinohara, A., Ogawa, H., and Ogawa, T. 1992. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell69: 457-470.

Smith, A.V. and Roeder, G.S. 1997. The yeast Red1 protein localizes to the cores of meiotic chromosomes. J. Cell Biol.136: 957-967.

Tsubouchi, H. and Ogawa, H. 1998. A novel mre11 mutation impairs processing of double-strand breaks of DNA during both mitosis and meiosis. Mol. Cell. Biol.18: 260-268.

Tsubouchi, H. and Roeder, G.S. 2002. The Mnd1 protein forms a complex with Hop2 to promote homologous chromosome pairing and meiotic double-strand break repair. Mol. Cell. Biol.22: 3078-3088.

-. 2003. The importance of genetic recombination for fidelity of chromosome pairing in meiosis. Dev. Cell5: 915-925.

-. 2004. The budding yeast Mei5 and Sae3 proteins act together with Dmc1 during meiotic recombination. Genetics168: 1219-1230.