Yeast RNA polymerase I enhancer is dispensable for transcription of the chromosomal rRNA gene and cell growth, and its apparent transcription enhancement from ectopic promoters requires Fob1 protein.

At the end of the 35S rRNA gene within ribosomal DNA (rDNA) repeats in Saccharomyces cerevisiae lies an enhancer that has been shown to greatly stimulate rDNA transcription in ectopic reporter systems. We found, however, that the enhancer is not necessary for normal levels of rRNA synthesis from chromosomal rDNA or for cell growth. Yeast strains which have the entire enhancer from rDNA deleted did not show any defects in growth or rRNA synthesis. We found that the stimulatory activity of the enhancer for ectopic reporters is not observed in cells with disrupted nucleolar structures, suggesting that reporter genes are in general poorly accessible to RNA polymerase I (Pol I) machinery in the nucleolus and that the enhancer improves accessibility. We also found that a fob1 mutation abolishes transcription from the enhancer-dependent rDNA promoter integrated at the HIS4 locus without any effect on transcription from chromosomal rDNA. FOB1 is required for recombination hot spot (HOT1) activity, which also requires the enhancer region, and for recombination within rDNA repeats. We suggest that Fob1 protein stimulates interactions between rDNA repeats through the enhancer region, thus helping ectopic rDNA promoters to recruit the Pol I machinery normally present in the nucleolus.

Interaction of Mre11 and Rad50: two proteins required for DNA repair and meiosis-specific double-strand break formation in Saccharomyces cerevisiae.

A temperature-sensitive mre11-1 mutation of Saccharomyces cerevisiae causes defects in meiotic recombination and DNA repair during vegetative growth at a restrictive temperature. We cloned the MRE11 gene and found that it encodes a 643-amino acid protein with a highly acidic region containing a heptad repeat of Asp at its C-terminus and is located downstream of YMR44 near the RNA1 locus on the right arm of chromosome XIII. Transcripts of the MRE11 gene increased transiently and showed the same kinetics as that of the RAD50 gene during meiosis. In a mre11 disruption mutant (mre11::hisG), meiosis-specific double-strand break (DSB) formation is abolished. A comparison of the properties of mre11::hisG and a rad50 deletion mutant (rad50 delta) indicated that both mutants exhibited similar phenotypes in both meiosis and mitosis. Characterization of two double mutants, mre11::hisG rad50 delta and mre11::hisG rad50S, showed that MRE11 and RAD50 belong to the same epistasis group with respect to meiotic DSB formation and mitotic DNA repair. Using a two-hybrid system, we found that Mre11 interacts with Rad50 and itself in vivo. These results suggest that Mre11 and Rad50 proteins work in a complex in DSB formation and DNA repair during vegetative growth.

Functions of the yeast meiotic recombination genes, MRE11 and MRE2.

Mutants defective in meiotic recombination were isolated using a disomic haploid strain of S. cerevisiae, and were classified into 11 genes. Two, MRE2 and MRE11, are new genes and nine are previously identified genes. The mre2 and mre11 deletion mutants are proficient in mitotic recombination, but are defective in meiotic recombination and in formation of viable spores. The spore inviability, however, is alleviated by an additional mutation, spo13, which bypasses meiosis I. In addition, neither meiosis specific DSBs at recombination hot-spots nor formation of synaptonemal complex occur in either mutant. Therefore, these two genes are involved in the formation of DSBs in meiotic recombination. While a temperature sensitive mre11-1 mutant is able to form DSBs at a permissive temperature, the formed DSBs are unable to resect at non permissive temperature. Therefore, the MRE11 gene is also involved in some step of the repair process after the DSB formation. Analysis of properties of the mre11 disruption mutant as well as the xrs2 mutant showed a similarity to those of the rad50 disruptant. We found that the mre11 disruption mutation is epistatic to rad50S mutation, as the xrs2 deletion mutation is epistatic to rad50S with regard to DSBs. Therefore, these three genes form an epistatic group. Interaction of the Mre11 protein with the Rad50 and the Xrs2 protein as well as alone was shown in vivo using the two-hybrid system. The MRE2 gene encodes a protein containing two sets of RRM. Deficiency of recombination in a mre2 mutant that has an amino acid substitution in the N-terminal RRM can be suppressed by the MER2 gene on the multicopy plasmid. Further analysis showed that the Mre2 protein is involved in meiosis-specific splicing of the MER2 transcripts in cooperation with the Mer1 protein. In conclusion, MRE genes are involved in the initiation of meiotic recombination through the formation of DSBs at recombination hot-spots in S. cerevisiae.