Biomedical subjects

B K Shafer

Publications and source records attributed to B K Shafer.

12 recordsLinked to original sources

PubMed2001

Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM1.

Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is approximately 100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an approximately 10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Delta strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.

DNA Damage↗

PubMed2000

The Saccharomyces cerevisiae DNA recombination and repair functions of the RAD52 epistasis group inhibit Ty1 transposition.

RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or delta) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA.

DNA Ligases↗

PubMed2000

The Saccharomyces cerevisiae RDN1 locus is sequestered from interchromosomal meiotic ectopic recombination in a SIR2-dependent manner.

Meiotic ectopic recombination occurs at similar frequencies among many sites in the yeast genome, suggesting that all loci are similarly accessible to homology searching. In contrast, we found that his3 sequences integrated in the RDN1 (rDNA) locus were unusually poor participants in meiotic recombination with his3 sequences at other sites. We show that the low rate of meiotic ectopic recombination resulted from the poor ability of RDN1::his3 to act as a donor sequence. SIR2 partially repressed interchromosomal meiotic ectopic recombination at RDN1, consistent with its role in regulating recombination, gene expression, and retrotransposition within RDN1. We propose that RDN1 is physically sequestered from meiotic homology searching mechanisms.

Alleles↗

PubMed1995

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 alpha. 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 alpha 1 (a transcriptional activator of alpha-specific genes) with Ste12p is believed to induce alpha-specific genes. We isolated and characterized a partially functional ste12 allele (ste12-T50), that is defective only in the activation of alpha-specific genes. ste12-T50 was isolated as a second-site mutation conferring the a mating phenotype on mat alpha 2 mutant cells. In mat alpha 2 cells, where due to the lack of repressor, alpha 2, both sets of cell-type-specific genes are expressed, ste12-T50 apparently tips the balance in favor of a-specific gene expression. Thus, mat alpha 2 ste12-T50 cells mate like a cells. Additional ste12 mutants that confer the a mating phenotype on mat alpha 2 cells have also been isolated.

Alleles↗

PubMed1995

DNA synthesis errors associated with double-strand-break repair.

Repair of a site-specific double-strand DNA break (DSB) resulted in increased reversion frequency for a nearby allele. Site-specific DSBs were introduced into the genome of Saccharomyces cerevisiae by the endonuclease encoded by the HO gene. Expression of the HO gene from a galactose-inducible promoter allowed efficient DNA cleavage at a single site in large populations of cells. To determine whether the DNA synthesis associated with repair of DSBs has a higher error rate than that associated with genome duplication, HO-induced DSBs were generated 0.3 kb from revertible alleles of trp1. The reversion rate of the trp1 alleles was approximately 100-fold higher among cells that had experienced an HO cut than among uninduced cells. The reverted allele was found predominantly on the chromosome that experienced the DNA cleavage.

Alleles↗

PubMed1994

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.

Amino Acid Sequence↗

PubMed1993

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.

Base Sequence↗

PubMed1992

Disruption of a silencer domain by a retrotransposon.

A galactose-inducible Ty element carrying the HIS3 gene has been used as an insertional mutagen to generate alpha-factor resistant mutants. This collection of Ty-induced mutations includes insertions into the gene for the alpha-factor receptor (STE2), several nonspecific STE genes, and mutations that lead to the expression of the normally silent HML alpha locus. The hml alpha "on" mutations fall into two classes, those that disrupt trans-acting regulators involved in silencing HML alpha and a novel class of mutations that activate HML alpha by insertion at that locus. The hml alpha::Ty "on" mutations illustrate the unusual ability of these retrotransposons to activate genes by overcoming gene silencing mechanisms. The hml alpha::Ty "on" mutations include examples of multimeric Ty arrays. Single Ty and solo delta insertion derivatives of these Ty multimers restore the ability of the silencing mechanism to repress HML alpha.

Blotting, Southern↗

PubMed1992

Ty element-induced temperature-sensitive mutations of Saccharomyces cerevisiae.

Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures. The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene. The hit4-1 mutation was located on chromosome XIII. These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response. The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein. Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family. In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus. The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes. The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome. This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein. The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.

Base Sequence↗

PubMed1990

Analysis of interchromosomal mitotic recombination.

A 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.

Alleles↗

PubMed1989

Analysis of the HO-cleaved MAT DNA intermediate generated during the mating type switch in the yeast Saccharomyces cerevisiae.

A galactose-inducible HO gene was used to induce mating type switching in heterothallic Saccharomyces cerevisiae cells arrested in G1, in rad52 mutants defective in DNA damage repair, and in cells lacking the donor cassettes. The HO-cleaved MAT intermediate is stable over significant lengths of time, i.e. HO cleavage is not coupled to the subsequent gene conversion event. The in vivo cleavage site was mapped to single base resolution by primer extension experiments on total genomic DNA. Cells arrested in G1 with alpha-factor switched mating type thus demonstrating that switches can occur in the absence of replication of the genome. rad52 mutants did not produce MAT DNA of the opposite mating type indicating that the block is prior to the gene duplication stage of the switch. In strains in which the HM donor cassettes are deleted the cut MAT DNA was degraded after induction of the HO gene.

Alleles↗

PubMed1988

Transposon tagging using Ty elements in yeast.

We have used the ability to induce high levels of Ty transposition to develop a method for transposon mutagenesis in Saccharomyces cerevisiae. To facilitate genetic and molecular analysis, we have constructed GAL1-promoted TyH3 or Ty917 elements that contain unique cloning sites, and marked these elements with selectable genes. These genes include the yeast HIS3 gene, and the plasmid PiAN7 containing the Tn903 NEO gene. The marked Ty elements retain their ability to transpose, to mutate the LYS2, LYS5, or STE2 genes, and to activate the promoterless his3 delta 4 target gene. Ty elements containing selectable genes are also useful in strain construction, in chromosomal mapping, and in gene cloning strategies.

Cloning, Molecular↗