Yeast cell cycle

Wittgenstein Award Z 39 Yeast cell cycle Kim A. NASMYTH 18.06.1999 The simultaneous separation of 46 pairs of sister chromatids at the metaphase to anaphase transition is one of the most dramatic events of the human cell cycle. Even as early ago as 1879, Flemming noticed that "the impetus causing nuclear threads to split longdituninally acts simultaneously on all of them". Chromosome splitting is an irreversible event and must therefore be highly regulated. Once sister chromatids separate from one another, damage to the genome cannot easily be repaired using recombination nor can mistakes in chromosome alignment be corrected. Sister chromatids are pulled to opposite halves of the cell by rnicrotubules emanating from spindle poles at opposite sides of the cell. One set of microtubules inter-digitates with others emanating from the opposite pole. Their role is to keep (and drive) the two poles apart. Meanwhile, a second set of microtubules attaches to chromosomes via specialized structures called kinetochores and pulls them towards the poles. Sister chromatids segregate away from each other because their kinetochores attach to microtubules emanating from opposite poles. Chromosomes are not mere passengers during this process. During metaphase, the tendency of microtubules to move sisters apart is counteracted by cohesion holding sisters together. Cohesion therefore generates the tension by which cells align sister chromatids on the metaphase plate. Were sisters to separate before spindle formation, it is difficult to imagine how cells could distinguish sisters from chromatids that were merely homologous. The sudden loss of cohesion, rather than an increase in the exertion of microtubules, is thought to trigger sister separation during anaphase. What holds sister chromatids together after chromosome replication, what is Flemming`s impetus that triggers loss of cohesion, and how do cells ensure that sister separation never occurs before all pairs of sister chromatids have been aligned on the metaphase plate? Such questions are equally pertinent to meiosis, where loss of sister chromatid cohesion within chromosome arms and centromeres must take place at different times. Genetic and biochemical studies on the budding yeast Saccharomyces cerevisiae have identified a multi-subunit complex called cohesin that is essential for holding sister chromatids together from DNA replication until the onset of anaphase. A related complex exists in human cells. In yeast, cohesin is loaded onto chromosomes during late G1 with the aid of a cohesin loading factor (CLF). Our working hypothesis is that connections between sister chromatids (mediated by cohesin) are established at replication forks with the aid of a protein called Eco1p. They persist until the onset of anaphase, whereupon activation of a "separin" protein (Esp1p) induces the proteolytic cleavage of the Scc1p cohesin subunit, which may be the trigger for sister chromatid separation. Separin is kept inactive from S phase till the onset of anaphase by its association with a securin protein (Pds1p). The liberation of separin from its securin is mediated by a multisubunit ubiquitin protein ligase called the Anaphase Promoting Complex or cyclosome (APC/C), which promotes the ubiquitination and hence proteolysis of securin. We are currently studying how CLF mediates the loading of cohesin onto chromosomes, what sort of structures are formed during this process, and how these structures are modified with the help of Eco1p during the passage of replication forks. We are also trying to establish whether separin is the protease responsible for cleaving Scc1p and are investigating how Scc1p cleavage remains tightly cell cycle regulated in mutants that lack securins. Loss of sister chromatid cohesion along chromosome arms is essential for chromosome segregation during meiosis I. Meanwhile, however, cohesion between sister centromeres persists so that it can later be used to align sisters on the meiosis II metaphase plate. The different timing of sister chromatid cohesion loss between chromosome arms and centromeres is therefore a crucial aspect of meiosis. The budding yeast genome encodes a second Scc1-like protein called Rec8p, which is needed for preventing precocious separation of sister chromatids during meiosis. Rec8p and other cohesin subunits are found all along the longditudinal axis of chromosomes during pachytene. They disappear from chromosome arms during the first meiotic division but persist in the neighbourhood of centromeres until metaphase II. We are currently investigating whether the removal of Rec8p from chromosomes is mediated by proteolytic cleavage induced by separin. We are also interested in understanding what prevents the removal of Rec8p from sequences close to centromeres until the second meiotic division.