Douglas Koshland

A Direct Link between Sister Chromatid Cohesion and Chromosome Condensation Revealed through the Analysis of MCD1 in S. cerevisiae

The S. cerevisiae MCD1 (mitotic chromosome determinant) gene was identified in genetic screens for genes important for chromosome structure. MCD1 is essential for viability and homologs are found from yeast to humans. Analysis of the mcd1 mutant and cell cycle-dependent expression pattern of Mcd1p suggest that this protein functions in chromosome morphogenesis from S phase through mitosis. The mcd1 mutant is defective in sister chromatid cohesion and chromosome condensation. The physical association between Mcd1p and Smc1p, one of the SMC family of chromosomal proteins, further suggests that Mcd1p functions directly on chromosomes. These data implicate Mcd1p as a nexus between cohesion and condensation. We present a model for mitotic chromosome structure that incorporates this previously unsuspected link.

Non-genetic individuality: chance in the single cell

The individuality of bacterial cells grown in homogeneous conditions was demonstrated by showing characteristic behavioural differences which persist over their lifespans. Poissonian fluctuation of small numbers of generator molecules can explain this individuality and may apply to such processes as differentiation and asynchrony of cultures.

Genome-Wide Mapping of the Cohesin Complex in the Yeast Saccharomyces cerevisiae

In eukaryotic cells, cohesin holds sister chromatids together until they separate into daughter cells during mitosis. We have used chromatin immunoprecipitation coupled with microarray analysis (ChIP chip) to produce a genome-wide description of cohesin binding to meiotic and mitotic chromosomes of Saccharomyces cerevisiae. A computer program, PeakFinder, enables flexible, automated identification and annotation of cohesin binding peaks in ChIP chip data. Cohesin sites are highly conserved in meiosis and mitosis, suggesting that chromosomes share a common underlying structure during different developmental programs. These sites occur with a semiperiodic spacing of 11 kb that correlates with AT content. The number of sites correlates with chromosome size; however, binding to neighboring sites does not appear to be cooperative. We observed a very strong correlation between cohesin sites and regions between convergent transcription units. The apparent incompatibility between transcription and cohesin binding exists in both meiosis and mitosis. Further experiments reveal that transcript elongation into a cohesin-binding site removes cohesin. A negative correlation between cohesin sites and meiotic recombination sites suggests meiotic exchange is sensitive to the chromosome structure provided by cohesin. The genome-wide view of mitotic and meiotic cohesin binding provides an important framework for the exploration of cohesins and cohesion in other genomes.

Cse4p Is a Component of the Core Centromere of Saccharomyces cerevisiae

Histones are fundamental structural components of chromatin and are expected to play important roles in chromosome dynamics. Here, we present direct evidence that Cse4p, a histone H3 variant, is a structural component of the core centromere of S. cerevisiae. In histone H4 and Cse4p mutants, the core centromere chromatin structure is disrupted at restrictive temperature. Overexpression of Cse4p suppresses this defect in the H4 mutant, implying that the two proteins act together in centromere structure. We show by chromatin immunoprecipitation experiments that Cse4p is specifically cross-linked to centromeric DNA. Furthermore, by immunofluorescence microscopy, Cse4p is found in discrete foci consistent with that expected for centromeres. These results suggest the kinetochore is assembled on a specialized centromeric nucleosome containing Cse4p.

A Molecular Determinant for the Establishment of Sister Chromatid Cohesion

Chromosome segregation, transcriptional regulation, and repair of DNA double-strand breaks require the cohesin protein complex. Cohesin holds the replicated chromosomes (sister chromatids) together to mediate sister chromatid cohesion. The mechanism of how cohesion is established is unknown. We found that in budding yeast, the head domain of the Smc3p subunit of cohesin is acetylated by the Eco1p acetyltransferase at two evolutionarily conserved residues, promoting the chromatin-bound cohesin to tether sister chromatids. Smc3p acetylation is induced in S phase after the chromatin loading of cohesin and is suppressed in G1 and G2/M. Smc3 head acetylation and its cell cycle regulation provide important insights into the biology and mechanism of cohesion establishment.

Sister Chromatid Cohesion: A Simple Concept with a Complex Reality

In eukaryotes, the process of sister chromatid cohesion holds the two sister chromatids (the replicated chromosomes) together from DNA replication to the onset of chromosome segregation. Cohesion is mediated by cohesin, a four-subunit SMC (structural maintenance of chromosome) complex. Cohesin and cohesion are required for proper chromosome segregation, DNA repair, and gene expression. To carry out these functions, cohesion is regulated by elaborate mechanisms involving a growing list of cohesin auxiliary factors. These factors control the timing and position of cohesin binding to chromatin, activate chromatin-bound cohesin to become cohesive, and orchestrate the orderly dissolution of cohesion. The 45-nm ringlike architecture of soluble cohesin is compatible with dramatically different mechanisms for both chromatin binding and cohesion generation. Solving the mechanism of cohesion and its complex regulation presents significant challenges but offers the potential to provide important insights into higher-order chromosome organization and chromosome biology.

Genetic analysis of the mitotic transmission of minichromosomes.

The fidelity of the mitotic transmission of minichromosomes in S. cerevisiae is monitored by a novel visual assay that allows one to detect changes in plasmid copy number in individual mitotic divisions. This assay is used to investigate the mitotic transmission of a plasmid containing a putative yeast origin of replication (ARS 1) and a centromere (CEN3). The rate of improper segregation for the minichromosome is 200-fold higher than observed for a normal chromosome. However, the replication of the minichromosome is stringently controlled; it overreplicates less than once per one thousand mitotic divisions. We also use this assay to isolate and characterize mutations in ARS 1 and CEN3. The mutations in ARS 1 define a new domain required for its optimal activity, and the mutations in CEN3 suggest that the integrity of element II is not essential for centromere function. Finally, the phenotypes of the mutations in ARS 1 and CEN3 are consistent with their function in replication and segregation, respectively.

RNase H and Multiple RNA Biogenesis Factors Cooperate to Prevent RNA:DNA Hybrids from Generating Genome Instability

Genome instability, a hallmark of cancer progression, is thought to arise through DNA double strand breaks (DSBs). Studies in yeast and mammalian cells have shown that DSBs and instability can occur through RNA:DNA hybrids generated by defects in RNA elongation and splicing. We report that in yeast hybrids naturally form at many loci in wild-type cells, likely due to transcriptional errors, but are removed by two evolutionarily conserved RNase H enzymes. Mutants defective in transcriptional repression, RNA export and RNA degradation show increased hybrid formation and associated genome instability. One mutant, sin3Δ, changes the genome profile of hybrids, enhancing formation at ribosomal DNA. Hybrids likely induce damage in G1, S and G2/M as assayed by Rad52 foci. In summary, RNA:DNA hybrids are a potent source for changing genome structure. By preventing their formation and accumulation, multiple RNA biogenesis factors and RNase H act as guardians of the genome.

The Centromeric Sister Chromatid Cohesion Site Directs Mcd1p Binding to Adjacent Sequences

Cohesion between sister chromatids occurs along the length of chromosomes, where it plays essential roles in chromosome segregation. We show here that the centromere, a cis-acting cohesion factor, directs the binding of Mcd1p, a cohesin subunit, to at least 2 kb regions flanking centromeres in a sequence-independent manner. The centromere is essential for the maintenance as well as the establishment of this cohesin domain. The efficiency of Mcd1p binding within the cohesin domain is independent of the primary nucleotide sequence of the centromere-flanking DNA but correlates with high A + T DNA content. Thus, the function of centromeres in the cohesion of centromere-proximal regions may be analogous to that of enhancers, nucleating cohesin complex binding over an extended chromosomal domain of A + T-rich DNA.

In vivo dissection of the chromosome condensation machinery reversibility of condensation distinguishes contributions of condensin and cohesin

The machinery mediating chromosome condensation is poorly understood. To begin to dissect the in vivo function(s) of individual components, we monitored mitotic chromosome structure in mutants of condensin, cohesin, histone H3, and topoisomerase II (topo II). In budding yeast, both condensation establishment and maintenance require all of the condensin subunits, but not topo II activity or phospho-histone H3. Structural maintenance of chromosome (SMC) protein 2, as well as each of the three non-SMC proteins (Ycg1p, Ycs4p, and Brn1p), was required for chromatin binding of the condensin complex in vivo. Using reversible condensin alleles, we show that chromosome condensation does not involve an irreversible modification of condensin or chromosomes. Finally, we provide the first evidence of a mechanistic link between condensin and cohesin function. A model discussing the functional interplay between cohesin and condensin is presented.