Brenda K. Shafer

Coconversion of Flanking Sequences with Homothallic Switching

Homothallic switching in S. cerevisiae involves replacing the DNA of the expressed allele at the mating type locus (MAT) with a duplicate of sequences from the unexpressed loci HML or HMR. The MATa and MAT alpha alleles differ by a DNA substitution that is flanked by sequences in common to MAT, and the donor loci HML and HMR. Using restriction site polymorphisms between MAT and the donor loci, we demonstrate that the extent of MAT DNA that is replaced during switching is variable and that there is a gradient of coconversion across the X region. Coconversion events occur on both sides of the double-strand cleavage by the HO gene product. The two cells produced after a switch often differ at the flanking site, indicating a DNA heteroduplex intermediate.

Mutations in the Saccharomyces cerevisiae RPB1 Gene Conferring Hypersensitivity to 6-Azauracil

RNA polymerase II (RNAPII) in eukaryotic cells drives transcription of most messenger RNAs. RNAPII core enzyme is composed of 12 polypeptides where Rpb1 is the largest subunit. To further understand the mechanisms of RNAPII transcription, we isolated and characterized novel point mutants of RPB1 that are sensitive to the nucleotide-depleting drug 6-azauracil (6AU). In this work we reisolated the rpo21-24/rpb1-E1230K allele, which reduces the interaction of RNAPII–TFIIS, and identified five new point mutations in RPB1 that cause hypersensitivity to 6AU. The novel mutants affect highly conserved residues of Rpb1 and have differential genetic and biochemical effects. Three of the mutations affect the “lid” and “rudder,” two small loops suggested by structural studies to play a central role in the separation of the RNA–DNA hybrids. Most interestingly, two mutations affecting the catalytic center (rpb1-N488D) and the homology box G (rpb1-E1103G) have strong opposite effects on the intrinsic in vitro polymerization rate of RNAPII. Moreover, the synthetic interactions of these mutants with soh1, spt4, and dst1 suggest differential in vivo effects.

Genetic Interactions of DST1 in Saccharomyces cerevisiae Suggest a Role of TFIIS in the Initiation-Elongation Transition

TFIIS promotes the intrinsic ability of RNA polymerase II to cleave the 3′-end of the newly synthesized RNA. This stimulatory activity of TFIIS, which is dependent upon Rpb9, facilitates the resumption of transcription elongation when the polymerase stalls or arrests. While TFIIS has a pronounced effect on transcription elongation in vitro, the deletion of DST1 has no major effect on cell viability. In this work we used a genetic approach to increase our knowledge of the role of TFIIS in vivo. We showed that: (1) dst1 and rpb9 mutants have a synthetic growth defective phenotype when combined with fyv4, gim5, htz1, yal011w, ybr231c, soh1, vps71, and vps72 mutants that is exacerbated during germination or at high salt concentrations; (2) TFIIS and Rpb9 are essential when the cells are challenged with microtubule-destabilizing drugs; (3) among the SDO (synthetic with Dst one), SOH1 shows the strongest genetic interaction with DST1; (4) the presence of multiple copies of TAF14, SUA7, GAL11, RTS1, and TYS1 alleviate the growth phenotype of dst1 soh1 mutants; and (5) SRB5 and SIN4 genetically interact with DST1. We propose that TFIIS is required under stress conditions and that TFIIS is important for the transition between initiation and elongation in vivo.

Recombination initiated by double-strand breaks

The HO endonuclease was used to introduce a site-specific double-strand break (DSB) in an interval designed to monitor mitotic recombination. The interval included the trp1 and his3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Mitotic recombination was monitored in a diploid carrying heteroalleles of trp1 and his3. The normal recognition sites for the HO endonuclease were mutated at the MAT alleles and a synthetic recognition site for HO endonuclease was placed between trp1 and his3 on one of the chromosomes. HO-induced cleavage resulted in efficient recombination in this interval. Most of the data can be explained by double-strand gap repair in which the cut chromosome acts as the recipient. However, analysis of some of the recombinants indicates that regions of heteroduplex were generated flanking the site of the cut, and that some recombinants were the result of the cut chromosome acting as the genetic donor.

Elevated Mutation Rate during Meiosis in Saccharomyces cerevisiae

Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis. Since the process of meiosis involves many programmed DSBs, we reasoned that this repair might also be mutagenic. Indeed, in the early 1960′s Magni and Von Borstel observed elevated reversion of recessive alleles during meiosis, and found that the revertants were more likely to be associated with a crossover than non-revertants, a process that they called “the meiotic effect.” Here we use a forward mutation reporter (CAN1 HIS3) placed at either a meiotic recombination coldspot or hotspot near the MAT locus on Chromosome III. We find that the increased mutation rate at CAN1 (6 to 21 –fold) correlates with the underlying recombination rate at the locus. Importantly, we show that the elevated mutation rate is fully dependent upon Spo11, the protein that introduces the meiosis specific DSBs. To examine associated recombination we selected for random spores with or without a mutation in CAN1. We find that the mutations isolated this way show an increased association with recombination (crossovers, loss of crossover interference and/or increased gene conversion tracts). Polζ appears to contribute about half of the mutations induced during meiosis, but is not the only source of mutations for the meiotic effect. We see no difference in either the spectrum or distribution of mutations between mitosis and meiosis. The correlation of hotspots with elevated mutagenesis provides a mechanism for organisms to control evolution rates in a gene specific manner.

Analysis of interchromosomal mitotic recombination

SummaryA novel synthetic locus is described that provides a simple assay system for characterizing mitotic recombinants. The locus consists of the TRP1 and HIS3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Defined trp1 and his3 alleles have been generated that allow the selection of interchromosomal recombinants in this interval. Trp+ or His+ recombinants can be divided into several classes based on coupling of the other alleles in the interval. The tight linkage of the CRY1 and MAT loci, combined with the drug resistance and cell type phenotypes that they respectively control, facilitates the classification of the recombinants without resorting to tetrad dissection. We present the distribution of spontaneous recombinants among the classes defined by this analysis. The data suggest that the recombination intermediate can have regions of symmetric strand exchange and that co-conversion tracts can extend over 1–3 kb. Continuous conversion tracts are favored over discontinuous tracts. The distribution among the classes defined by this analysis is altered in recombinants induced by UV irradiation.

A genetic assay for transcription errors reveals multilayer control of RNA polymerase II fidelity.

We developed a highly sensitive assay to detect transcription errors in vivo. The assay is based on suppression of a missense mutation in the active site tyrosine in the Cre recombinase. Because Cre acts as tetramer, background from translation errors are negligible. Functional Cre resulting from rare transcription errors that restore the tyrosine codon can be detected by Cre-dependent rearrangement of reporter genes. Hence, transient transcription errors are captured as stable genetic changes. We used this Cre-based reporter to screen for mutations of Saccharomyces cerevisiae RPB1 (RPO21) that increase the level of misincorporation during transcription. The mutations are in three domains of Rpb1, the trigger loop, the bridge helix, and in sites involved in binding to TFIIS. Biochemical characterization demonstrates that these variants have elevated misincorporation, and/or ability to extend mispaired bases, or defects in TFIIS mediated editing.

Isolation, identification and characterization of the FUN12 gene of Saccharomyces cerevisiae.

We have cloned and characterized the FUN12 gene which is found on chromosome 1 of Saccharomyces cerevisiae. The complete nucleotide (nt) sequence of the cDNA and the genomic clones shows that FUN12 is expressed as a 3.7-kb message and should encode a 97 kDa-protein. Immunoprecipitations using antipeptide antibodies showed that the cells contain a Fun12p of this size. The databases contain no nt sequences that are homologous to FUN12 and no protein homologous to Fun12p. Gene disruption experiments showed that FUN12 is an essential gene.

A ste12 allele having a differential effect on a versus alpha cells.

The transcriptional activator Ste12p is a key component of the yeast pheromone response pathway: phosphorylated as a consequence of signal transduction, it activates transcription of genes that promote mating and the subsequent fusion of the two cell types a and α. Activation by Ste12p requires three types of protein-protein interaction between DNA-binding activator proteins: (1) Ste12p by itself can induce non-cell-type-specific genes involved in mating; (2) cooperation of the transactivator Mcm1p with Ste12p induces a-specific genes; and (3) formation of a complex of the activator proteins Mcm1p and α1 (a transcriptional activator of α-specific genes) with Ste12p is believed to induce α-specific genes. We isolated and characterized a partially functional ste12 allele (ste12-T50), that is defective only in the activation of α-specific genes. ste12-T50 was isolated as a second-site mutation conferring the a mating phenotype on matα2 mutant cells. In matα2 cells, where due to the lack of repressor, α2, both sets of cell-type-specific genes are expressed, ste12-T50 apparently tips the balance in favor of a-specific gene expression. Thus, matα2 ste12-T50 cells mate like a cells. Additional ste12 mutants that confer the a mating phenotype on matα2 cells have also been isolated.