The 2 micron plasmid purloins the yeast cohesin complex: a mechanism for coupling plasmid partitioning and chromosome segregation?

The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locus. The periodicity of cohesin association and dissociation is nearly identical for the plasmid and the chromosomes. The timely disassembly of cohesin is a prerequisite for plasmid segregation. Cohesin-mediated pairing and unpairing likely provides a counting mechanism for evenly partitioning plasmids either in association with or independently of the chromosomes

The Exact Critical Bubble Free Energy and the Effectiveness of Effective Potential Approximations

To calculate the temperature at which a first-order cosmological phase transition occurs, one must calculate $F_c(T)$, the free energy of a critical bubble configuration. $F_c(T)$ is often approximated by the classical energy plus an integral over the bubble of the effective potential; one must choose a method for calculating the effective potential when $V''<0$. We test different effective potential approximations at one loop. The agreement is best if one pulls a factor of $\mu^4/T^4$ into the decay rate prefactor [where $\mu^2 = V''(\phi_f)$], and takes the real part of the effective potential in the region $V''<0$. We perform a similar analysis on the 1-dimensional kink.Comment: 11 pages plus 3 figures in jyTeX; CALT-68-188

Four Novel Suppressors of gic1 gic2 and Their Roles in Cytokinesis and Polarized Cell Growth in Saccharomyces cerevisiae

Gic1 and Gic2 are two Cdc42/Rac interactive binding (CRIB) domain-containing effectors of Cdc42-GTPase that promote polarized cell growth in S. cerevisiae. To identify novel genes that functionally interact with Gic1 and Gic2, we screened for high-copy suppressors of a gic1 gic2 temperature-sensitive strain. We identified two pairs of structurally related genes, SKG6-TOS2 and VHS2-MLF3. These genes have been implicated in polarized cell growth, but their functions have not previously been characterized. We found that overproduction of Skg6 and Tos2 in wild-type cells causes aberrant localization of Cdc3 septin and actin structures as well as defective recruitment of Hof1 and impaired formation of the septum at the mother-bud neck. These data suggest a negative regulatory function for Skg6 and Tos2 in cytokinesis. Consistent with this model, deletion of SKG6 suppresses the growth defects associated with loss of HOF1, a positive regulator of cytokinesis. Our analysis of the second pair of gic1 gic2 suppressors, VHS2 and MLF3, suggests that they regulate polarization of the actin cytoskeleton and cell growth and function in a pathway distinct from and parallel to GIC1 and GIC2

Mcm1 Promotes Replication Initiation by Binding Specific Elements at Replication Origins

Minichromosome maintenance protein 1 (Mcm1) is required for efficient replication of autonomously replicating sequence (ARS)-containing plasmids in yeast cells. Reduced DNA binding activity in the Mcm1-1 mutant protein (P97L) results in selective initiation of a subset of replication origins and causes instability of ARS-containing plasmids. This plasmid instability in the mcm1-1 mutant can be overcome for a subset of ARSs by the inclusion of flanking sequences. Previous work showed that Mcm1 binds sequences flanking the minimal functional domains of ARSs. Here, we dissected two conserved telomeric X ARSs, ARS120 (XARS6L) and ARS131a (XARS7R), that replicate with different efficiencies in the mcm1-1 mutant. We found that additional Mcm1 binding sites in the C domain of ARS120 that are missing in ARS131a are responsible for efficient replication of ARS120 in the mcm1-1 mutant. Mutating a conserved Mcm1 binding site in the C domain diminished replication efficiency in ARS120 in wild-type cells, and increasing the number of Mcm1 binding sites stimulated replication efficiency. Our results suggest that threshold occupancy of Mcm1 in the C domain of telomeric ARSs is required for efficient initiation. We propose that origin usage in Saccharomyces cerevisiae may be regulated by the occupancy of Mcm1 at replication origins

Identification of Novel, Evolutionarily Conserved Cdc42p-interacting Proteins and of Redundant Pathways Linking Cdc24p and Cdc42p to Actin Polarization in Yeast

In the yeast Saccharomyces cerevisiae, Cdc24p functions at least in part as a guanine-nucleotide-exchange factor for the Rho-family GTPase Cdc42p. A genetic screen designed to identify possible additional targets of Cdc24p instead identified two previously known genes, MSB1 and CLA4, and one novel gene, designated MSB3, all of which appear to function in the Cdc24p–Cdc42p pathway. Nonetheless, genetic evidence suggests that Cdc24p may have a function that is distinct from its Cdc42p guanine-nucleotide-exchange factor activity; in particular, overexpression of CDC42 in combination with MSB1 or a truncated CLA4 in cells depleted for Cdc24p allowed polarization of the actin cytoskeleton and polarized cell growth, but not successful cell proliferation. MSB3 has a close homologue (designated MSB4) and two more distant homologues (MDR1 and YPL249C) in S. cerevisiae and also has homologues in Schizosaccharomyces pombe, Drosophila (pollux), and humans (the oncogene tre17). Deletion of either MSB3 or MSB4 alone did not produce any obvious phenotype, and the msb3 msb4 double mutant was viable. However, the double mutant grew slowly and had a partial disorganization of the actin cytoskeleton, but not of the septins, in a fraction of cells that were larger and rounder than normal. Like Cdc42p, both Msb3p and Msb4p localized to the presumptive bud site, the bud tip, and the mother-bud neck, and this localization was Cdc42p dependent. Taken together, the data suggest that Msb3p and Msb4p may function redundantly downstream of Cdc42p, specifically in a pathway leading to actin organization. From previous work, the BNI1, GIC1, and GIC2 gene products also appear to be involved in linking Cdc42p to the actin cytoskeleton. Synthetic lethality and multicopy suppression analyses among these genes, MSB, and MSB4, suggest that the linkage is accomplished by two parallel pathways, one involving Msb3p, Msb4p, and Bni1p, and the other involving Gic1p and Gic2p. The former pathway appears to be more important in diploids and at low temperatures, whereas the latter pathway appears to be more important in haploids and at high temperatures