Mammalian cell cycle regulation : characterisation of three genes expressed in proliferating cells

Sammanfattning: MAMMALIAN CELL CYCLE REGULATION: CHARACTERISATION OF THREE GENES EXPRESSED IN PROLIFERATING CELLS Maria Starborg Laboratory of Moleeular Genetics, Department of Cell and Moleeular Biology Karolinska Institutet, S-171 77 Stockholm Proliferating cells undergo different phases in their life cycle. During interphase thecells grow and duplicate their DNA content, followed by a cell division phase, mitosis,which results in two daughter cells. The transitions between the different events of the cellcycle are strictly regulated by a checkpoint machinery to ensure the integrity of the DNA.This control system checks that an early event is completed before a later event is initiated.For example, the DNA replication must be completed before a cell can enter mitosis. Thefailure of any of the checkpoints eould lead to uneontrolled eell proliferation and theaccumulation of genetic damage. It is therefore of great interest to identify and characterisenew genes that are involved in the control of the cell cycle. This thesis presents the isolation and characterisation of three murine genes, tsg23,tsg24 and tsgl26, all expressed in proliferating cells. The first gene described, tsg23, was shown to be the murine homologue, Pl, of theyeast MCM3 gene, which has been shown to be important for the initiation of DNAreplication. The MCM3 protein belongs to a protein family suggested to function asreplication licensing factors. The Pl protein is located in the nuclei during all stages ofinterphase and in the extrachromosomal regions during mitosis. The P1 protein accumulatesin the heterochromatin regions before replication, an association which is lost during the Sphase. Our results suggest that the Pl protein could be involved in the regulation of theinitiation of DNA replication. In contrast to MCM3, we found that P1 remains loealised inthe nuclei throughout interphase. This rises the possibility that the accumulation of themammalian MCM proteins are regulated by a different mechanism than theirs yeastcounterparts. An alternative explanation is that the activity of the yeast MCM proteinscould be regulated by two different mechanisms. The second gene described, tsg24, possibly represents the murine homologue of theAspergillus nidulans BimE gene. It has been suggested that the BimE protein functions as acell cycle checkpoint regulator, which controls the transition between different checkpoints.The Tsg24 protein is a nuclear protein located in the centromere regions of thechromosomes, chromosomal regions important for the proper segregation of sisterchromatids during mitosis. The centromeric localisation of the Tsg24 protein most probablyexplains one of the observed BimE phenotypes, the metaphase arrest, as inactivation ofother centromere proteins also leads to a metaphase arrest. More intriguing is the secondphenotype displayed by BimE mutants, premature chromosome condensation. However, itis possible that some centromere proteins also may be involved in the regulation ofchromosome condensation. Interestingly, another centromere protein, CENP-D, is related toRCC1, a protein also known to regulate chromosome condensation. The third gene investigated, tsgl26, was found to be the murine homologue of thehuman Ki-67 gene. The Ki-67 protein is exclusively expressed in proliferating cells and istherefore used as a clinical marker to assess the proliferative capacity of tumour cells. Boththe Tsgl26 and Ki-67 proteins contain repetitive sequences containing several conservedmotifs, including one motif observed in proteins which interact with DNA. The Tsgl26protein begins to accumulate in the nucleus at the beginning of the G1 phase. During the Sand the G2 phases, the Tsgl26 protein becomes localised to the nucleolus and to theheteroehromatin regions. At the onset of mitosis, the Tsgl26 protein is redistributed andassociates with the mitotie chromosomes. The intracellular distribution of the Tsgl26protein during the cell cycle suggests that it could have a chromatin-associated function inboth interphase and mitotic cells. Microinjection of anti-Tsgl26 antibodies intoproliferating cells delays cell cyele progression, indieating that the Tsgl26 protein has anessential nuclear function.Key words: Cell cycle, DNA replication, licensing factor, mitosis, ceheckpoint regulator,centromere regions, cell proliferation marker, nucleolar protein, spermatogenesisISBN 91-628-1847-3 Stockholm 1995