The role of cohesins and the APC-ESP1 in regulating anaphase in vertebrate cells

In the eukaryotic cell cycle, duplication and segregation of the genome occcurs in two temporally distinct steps, DNA replication in S phase and chromosome segregation in anaphase. Between these two events, replicated DNA molecules ("sisters") remain physically connected, a phenomenon called cohesion. Cohesion is required for the formation of bilaterally symmetric chromosomes that are composed of two sister chromatids and thus enables the symmetric segregation of chromatids once a spindle apparatus has formed. Defects in sister chromatid cohesion could lead to unequal chromosome segregation and thus to the formation of aneuploid daughter cells. Aneuploidy is frequently observed in human tumor cells and also in genetic diseases such as trisomy 21. Analyzing the molecular basis of cohesion win therefore be important both for understanding the mechanisms of mitosis and meiosis and to obtain insight into the etiology of human diseases associated with aneuploidy. How cohesion is established during DNA replication, maintained until metaphase and dissolved at the metaphase- anaphase transition is only poorly understood. Recent experiments in yeast and Xenopus indicate that chromosomal proteins called cohesins are required to physically hold sister chromatids together and that their dissociation from chromosomes is a prerequisite for anaphase. Several observations suggest that activation of a multi-subunit ubiquitination complex, called the anaphase-promoting complex (APC) or cyclosome, is required for this event, but how APC activation initiates anaphase is only poorly understood. The long term goal of the research proposed here is to obtain insight into how mitotic activation of the APC initiates sister chromatid separation in vertebrate cells. To achieve this goal we propose the following aims: 1. We will analyze the function of a protein called ESP1 in human cells and in Xenopus cell cycle extracts because recent genetic experiments indicate that the yeast homolog of this protein initiates anaphase following APC activation. 2. To analyze the mechanism that triggers the dissociation of cohesins from chromosomes prior to anaphase we will use biochemical fractionation and in vitro cohesin dissociation assays to identify mitotic activators of this event. We will also test whether the APC and ESP1 are required for mitotic dissociation of cohesins from chromatin. 3. To obtain insight into how cohesins organize chromatin and may physically connect sister chromatids we will purify complexes composed of cohesins and will analyze their low resolution structure by electron microscopy. We will further attempt to reconstitute such complexes from recombinant subunits. 4. To obtain insight into the spatial and temporal regulation of cohesins, and of other chromosomal proteins that may also play a role in controlling anaphase we will follow the intracellular distribution of these proteins in mitotic cells by immunolocalization techniques.

Most organisms inherit their genomes from one ceIl generation to the next by first duplicating their DNA during S- phase of the celi cycle and then segregating it into two separate copies ciuring mitosis. In mitosis, DNA is packaged into chromosomes in which the two replicated DNA molecules can be microscopically seen as sister chromatids. Each chromosome is then captured by two opposing poles of the spindle which allows the segregation of sister chromatids towards opposite poles during a phase of mitosis that is called anaphase. The aim of this project was to understand how sister chromatids are separated during mitosis in human cells. We discovered that this process is mediated by a special enzyme that cleaves proteins that are holding sister chromatids together. We called this enzyme separase because it physically separates sister chromatjds at the onset of anaphase. Through most of the ceII cycle separase is kept inactive by an inhibitory protein called securin. Once all chromosomes have been attached to both poles of the mitotic spindle securin is destroyed, leading to activation of separase and subsequently to sister chromatid separation in anaphase. The missegregation of chromosomes can cause congenital diseases such as Down syndrome (trisomy 21) and may contribute to the formation of human cancers. Understanding the regulation of chromosome segregation may therefore help to elucidate defects that cause these diseases. Regulators of chromosome segregation such as separase may furthermore represent potential drug targets whose pharmacological inhibition might be of therapeutic benefit for cancer patients.