Carl Mann

Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss

A colony color assay that measures chromosome stability is described and is used to study several parameters affecting the mitotic maintenance of yeast chromosomes, including ARS function, CEN function, and chromosome size. A cloned ochre-suppressing form of a tRNA gene, SUP11, serves as a marker on natural and in vitro-constructed chromosomes. In diploid strains homozygous for an ochre mutation in ade2, cells carrying no copies of the SUP11 gene are red, those carrying one copy are pink, and those carrying two or more copies are white. Thus, the degree of red sectoring in colonies reflects the frequency of mitotic chromosome loss. The assay also distinguishes between chromosome loss (1:0 segregation) and nondisjunction (2:0 segregation). The most dramatic effect on improving mitotic stability is caused by increasing chromosome size. Circular chromosomes increase in stability through a size range up to approximately 100 kb, but do not continue to be stabilized above this value. However, linear chromosomes continue to increase in mitotic stability throughout the size range tested (up to 137 kb). It is possible that the mitotic stability of linear chromosomes is proportional to chromosome length, up to a plateau value that has not yet been reached in our synthetic constructions.

Civ1 (CAK In Vivo), a Novel Cdk-Activating Kinase

Cyclin-dependent protein kinases (Cdks) play key roles in regulating cell division and gene expression. Most Cdks require binding of a cyclin and phosphorylation by a Cdk-activating kinase (CAK) to be active. We report the identification of Civ1 (CAK in vivo), a novel CAK activity in S. cerevisiae. Civ1 is most similar in sequence to the Cdks, but unlike them is active as a monomer and may thus be the founding member of a novel family of kinases. Civ1 binds tightly to and phosphorylates Cdc28, thereby allowing its subsequent activation by the binding of a cyclin. The CIV1 gene is essential for yeast cell viability, and Cdc28 phosphorylation and activity are conditionally inhibited in a civ1-4 temperature-sensitive mutant. Civ1 is the only CAK for which there are genetic data indicating that its activity is physiologically relevant in vivo.

Centromeric DNA from Saccharomyces cerevisiae

We have isolated 29 kilobase-pairs of DNA encompassing the centromere-linked gene, TRP1, of Saccharomyces cerevisiae. Two DNA sequences within the isolated region, ARS1 and ARS4, allow autonomous replication of chimeric DNA molecules upon introduction into yeast. Yeast strains transformed by ARS-containing DNA molecules are unstable; they lose the transformed phenotype and, concomitantly, the hybrid molecules at high frequency. Another function, CEN4, stabilizes the ARS-containing hybrids. CEN4 allows proper segregation of hybrid molecules during mitosis and meiosis. Like its predecessor. CEN3 (Clarke & Carbon, 1980b). CEN4 behaves genetically like a yeast centromere. The centromere-linked sequences we isolated from chromosome IV of yeast are not highly repeated and contain transcriptionally active DNA sequences. We have also isolated other CEN sequences using an enrichment procedure based on the enhanced mitotic stability of molecules carrying both CEN and ARS. Together, the ARS and CEN functions allow DNA molecules to predominantly behave as independent yeast linkage groups. However, improper disjunction of these ARS-CEN hybrids occurs in 3 to 14% of the mitotic cell divisions and ⩽3% of the meiotic divisions while the segregation of other yeast chromosomes, including chromosome IV, is normal. No more than 2% of these monovalent circular chromosomes undergo precocious segregation of sister chromatids during the first meiotic division rather than the second. Thus functional yeast chromosomes have been reconstructed by piecing together fragments of centromeric DNA with fragments that allow autonomous replication in yeast.

Structural basis for the interaction of Asf1 with histone H3 and its functional implications

Asf1 is a conserved histone chaperone implicated in nucleosome assembly, transcriptional silencing, and the cellular response to DNA damage. We solved the NMR solution structure of the N-terminal functional domain of the human Asf1a isoform, and we identified by NMR chemical shift mapping a surface of Asf1a that binds the C-terminal helix of histone H3. This binding surface forms a highly conserved hydrophobic groove surrounded by charged residues. Mutations within this binding site decreased the affinity of Asf1a for the histone H3/H4 complex in vitro, and the same mutations in the homologous yeast protein led to transcriptional silencing defects, DNA damage sensitivity, and thermosensitive growth. We have thus obtained direct experimental evidence of the mode of binding between a histone and one of its chaperones and genetic data suggesting that this interaction is important in both the DNA damage response and transcriptional silencing.

Sgt1p contributes to cyclic AMP pathway activity and physically interacts with the adenylyl cyclase Cyr1p/Cdc35p in budding yeast.

ABSTRACT Sgt1p is a highly conserved eucaryotic protein that is required for both SCF (Skp1p/Cdc53p-Cullin-F-box)-mediated ubiquitination and kinetochore function in yeast. We show here that Sgt1p is also involved in the cyclic AMP (cAMP) pathway in Saccharomyces cerevisiae. SGT1 is an allele-specific suppressor of cdc35-1, a thermosensitive mutation in the leucine-rich repeat domain of the adenylyl cyclase Cyr1p/Cdc35p. We demonstrate that Sgt1p and Cyr1p/Cdc35p physically interact and that the activity of the cAMP pathway is affected in an sgt1 conditional mutant. Sequence analysis suggests that Sgt1p has features of a cochaperone. Thus, Sgt1p is a novel activator of adenylyl cyclase in S. cerevisiae and may function in the assembly or the conformational activation of specific multiprotein complexes.

Yeast homolog of a cancer‐testis antigen defines a new transcription complex

We have isolated a new yeast gene (PCC1) that codes for a factor homologous to human cancer‐testis antigens. We provide evidence that Pcc1p is a new transcription factor and that its mutation affects expression of several genes, some of which are involved in cell cycle progression and polarized growth. Mutation of Pcc1p also affects the expression of GAL genes by impairing the recruitment of the SAGA and Mediator co‐activators. We characterize a new complex that contains Pcc1p, a kinase, Bud32p, a putative endopeptidase, Kae1p and two additional proteins encoded by ORFs YJL184w and YMLO36w. Genetic and physical interactions among these proteins strongly suggest that this complex is a functional unit. Chromatin immunoprecipitation experiments and multiple genetic interactions of pcc1 mutants with mutants of the transcription apparatus and chromatin modifying enzymes underscore the direct role of the complex in transcription. Functional complementation experiments indicate that the transcriptional function of this set of genes is conserved throughout evolution.

RPC40, a Unique Gene for a Subunit Shared between Yeast RNA Polymerases A and C

Yeast RNA polymerases A and C share an approximately equal to 40 kd subunit. We have identified, sequenced, and mutagenized in vitro the AC40 subunit gene. The RPC40 gene is unique in the yeast genome and is required for cell viability. This gene contains an open reading frame encoding a 37.6 kd protein having no significant homology with bacterial RNA polymerase subunits. The promoter region contains a 19 bp sequence also present in the largest subunit of RNA polymerase C. It also contains a well-conserved RPG box, a sequence found in the promoter region of many genes encoding the translational apparatus. A novel, plasmid-shuffling method was developed to isolate a large number of RPC40 ts mutants. One of these, ts4, was shown to be defective in the synthesis of RNA polymerases A and C at the restrictive temperature. In contrast, RNA polymerase B was made normally.

The histone chaperone Asf1 at the crossroads of chromatin and DNA checkpoint pathways

Nucleosome assembly involves deposition of a heterotetramer of histones H3/H4 onto DNA followed by two heterodimers of histones H2A/H2B. Cycles of nucleosome assembly and disassembly are essential to cellular events such as replication, transcription, and DNA repair. After synthesis in the cytoplasm, histones are shuttled into the nucleus where they are associated with chaperone proteins. Chaperones of histones H3/H4 include CAF-I, the Hir proteins, and Asf1. CAF-I and the Hir proteins function as replication-coupled and replication-independent deposition factors for H3/H4, respectively, whereas Asf1 may play a role in both pathways. In addition to acting as assembly factors, histone chaperones assist nucleosome dissociation from DNA and they may recruit other proteins to chromatin. The past few years have witnessed a notable accumulation of genetic, biochemical, and structural data on Asf1, which motivated this review. We discuss the sequence and structural features of Asf1 before considering its roles in nucleosome assembly/disassembly, the cellular response to DNA damage, and the regulation of gene expression. We emphasize the key role of Asf1 as a central node in a network of partners that place it at the crossroads of chromatin and DNA checkpoint pathways.

Yaf9, a novel NuA4 histone acetyltransferase subunit, is required for the cellular response to spindle stress in yeast.

ABSTRACT Yaf9 is one of three proteins in budding yeast containing a YEATS domain. We show that Yaf9 is part of a large complex and that it coprecipitates with three known subunits of the NuA4 histone acetyltransferase. Although Esa1, the catalytic subunit of NuA4, is essential for viability, we found that yaf9Δ mutants are viable but hypersensitive to microtubule depolymerizing agents and synthetically lethal with two different mutants of the mitotic apparatus. Microtubules depolymerized more readily in the yaf9Δ mutant compared to the wild type in the presence of nocodazole, and recovery of microtubule polymerization and cell division from limiting concentrations of nocodazole was inhibited. Two other NuA4 mutants (esa1-1851 and yng2Δ) and nonacetylatable histone H4 mutants were also sensitive to benomyl. Furthermore, wild-type budding yeast were more resistant to benomyl when grown in the presence of trichostatin A, a histone deacetylase inhibitor. These results strongly suggest that acetylation of histone H4 by NuA4 is required for the cellular resistance to spindle stress.

Silent repair accounts for cell cycle specificity in the signaling of oxidative DNA lesions

Reactive oxygen species are the most important source of DNA lesions in aerobic organisms, but little is known about the activation of the DNA checkpoints in response to oxidative stress. We show that treatment of yeast cells with sublethal concentrations of hydrogen peroxide induces a Mec1‐dependent phosphorylation of Rad53 and a Rad53‐dependent cell cycle delay specifically during S phase. The lack of Rad53 phosphorylation after hydrogen peroxide treatment in the G1 and G2 phases is due to the silent repair of oxidative DNA lesions produced at these stages by the base excision repair (BER) pathway. Only the disruption of the BER pathway and the accumulation and/or treatment of DNA intermediates by alternative repair pathways reveal the existence of primary DNA lesions induced at all phases of the cell cycle by hydrogen peroxide. Our data illustrate both the concept of silent repair of DNA damage and the high sensitivity of S‐phase cells to hydrogen peroxide.