Analysis of the S. pombe sister chromatid cohesin subunit in response to DNA damage agents during mitosis.

Q Science (General);R Medicine (General)

Bhatti, Saeeda ; McInerny, Christopher

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: S. pombe, cohesins, mitotis, meiosis, MMS, phleomycin, UV, northerns, PFGE;   

Others  :  http://theses.gla.ac.uk/292/1/2008BhattipHd.pdf

来源: University of Glasgow

【 摘 要 】

The accurate distribution of a fully replicated, intact genome perhaps defines the purpose of the cell cycle, which is a complex, organized series of events pertaining to the production of daughter cells.Every eukaryotic cell undergoes two alternative life cycles mitosis or meiosis, the first producing identical daughter cells of a diploid genetic content, and the second for genetic diversity and cells of a haploid genetic content.Studies in various experimental systems, from yeasts to humans, have identified the conservation of many basic features, and thus produced a unified view that this process is essentially the same in all eukaryotes (Nurse, 2000).The relevance of cell cycle studies cannot be overstated and their importance in elucidating the underlying causes of many diseases has proved invaluable in modern medicine.Chromosomes can be perceived as the packages for genetic information; they are replicated during S phase in both mitosis and meiosis to produce two identical sister chromatids which are subsequently segregated during the respective anaphases of both life cycles.Cohesin is a proteinacious structural complex holding sister chromatids together until nuclear division.Defects in this complex can cause chromosome mis-segregation inevitably causing an unbalanced genetic cell content which can either manifest as disease or give rise to inviable progeny.The subjects of this study are two homologous cohesin genes rad21+ and rec8+ expressed exclusively in either mitosis or meiosis respectively of the fission yeast Schizosaccharomyces pombe.All eukaryotes have two copies of these genes both analogously expressed and functionally similar, but undergoing life cycle specific expression.The aim of this study was to investigate the mitotic and meiotic specific functions of rad21+ and rec8+.To this end their specific pattern of expression was exchanged so that rad21+ was expressed in the meiotic cycle and rec8+ in the mitotic cycle.Previous work from the laboratory showed that rec8+ when expressed in mitosis at G1-S instead of rad21+ allows normal growth and division.Despite this initial observation, when the effects of various damaging agents applied during mitosis were examined some significant differences became apparent.In comparison to wild-type cells with functional SpRad21p, cells in which SpRec8p was expressed showed a reduction in repair efficiency which was revealed from cell survival, gene expression and chromosome integrity data. The first results chapter of this thesis introduces the two fission yeast cohesin subunits of interest. Firstly, the similarities between SpRec8p and SpRad21p are highlighted in terms of cell cycle regulation via the control of the DSC1 complex, and their similar functions to prevent premature separation of sister chromatids during anaphase.Secondly, the main differences between the two cohesins are emphasised, in that both are solely expressed during alternative life cycles.During the course of a synchronous wild-type mitosis, northern analysis of rad21+ mRNA levels revealed that this cohesin subunit is periodically expressed with peak transcript levels present at the G1-S boundary whilst, in contrast, no rec8+ transcript was detected throughout mitosis.For the duration of a synchronous meiotic cycle, peak levels of rec8+ transcript were detected, again at the G1-S boundary, whereas no rad21+ transcript was detectable.These data are consistent with and confirm published observations (Lin et al., 1992; Birkenbihl & Subramani, 1995). Following these preliminary experiments, a more in depth analysis of the cohesin subunits was completed, utilising three strains: a wild-type control, a rad21-45 mutant, and a repressible strain, rad21P:rec8+ nmt1P:rad21+.In the latter strain cohesin expression is swapped, thus forcing expression of the meiotic cohesin rec8+ in the mitotic cell cycle and replacing rad21+.The strains were treated with three damaging agents, ultraviolet-C(UV-C) radiation, methyl-methane sulfonate (MMS) and phleomycin.Qualitative and quantitative data were collected which demonstrated that regardless of the damaging agent used, increasing concentrations resulted in decreased cell survival in the tested strains.With UV-C most cell death took place at 150 J, decrease in survival rates for MMS treated cells was more apparent at a concentration of 0.3%, and the phleomycin data showed most cell death at 20 μgml-1.Furthermore, when the survival rates of the three strains were compared between the different damaging agents the pattern of survival was consistently in the order of the wild-type control most resistant, followed by the rad21-45 mutant, and the rad21P:rec8+ nmt1P:rad21+ strain most sensitive.These data allowed a visual analysis of the strains, demonstrating that the strain in which rec8+ was expressed had the least viability irrespective of the damaging agent.The effects of MMS were analysed further in the three strains with quantification of transcriptional profiles of the two cohesins by the northern blot technique and analysis of chromosome integrity by pulse-field gel electrophoresis (PFGE).The northern blot experiments indicated that rad21+ expression levels increased with higher concentrations of MMS, and cell viability decreased at concentrations above 0.1%.The induction of rad21+ transcription after addition of MMS was also observed in cells arrested at G2-M after just 60 minutes, implying that this DNA damaging agent causes the direct induction of expression of this cohesin gene. Levels of rec8+ transcript were also apparent in the rad21P:rec8+ nmt1P:rad21+ strain at the same MMS concentration, although expression was not induced.Chromosome integrity experiments were carried out using PFGE at a concentration of 0.05% MMS.Samples for PFGE and microscopic analysis were taken before, during and after MMS treatment.Through the course of the experiments samples were taken hourly to monitor growth, which demonstrated that after recovery wild-type cells resumed normal growth; similarly, the rad21-45 mutant cells returned to an almost normal growth rate. However, the strain in which rec8+ was expressed instead of rad21+ did not recover a normal growth pattern.Although northern data revealed the presence of rec8+ transcript at a concentration of 0.1%, PFGE data suggested that repair to chromosomes did not occur.Microscopic analysis revealed that only wild-type was unaffected, with the other two strains demonstrating elongated phenotypes characteristic of cell division cycle arrested cells.PFGE also revealed that after the recovery period, the only strain to recover the three chromosomes was wild-type.Thus, data presented in this thesis suggest that the basic growth functions of rec8+ and rad21+ are conserved between the two genes during mitosis and meiosis, but that mitotic DNA repair is specific to rad21+.This research offers new insights in the function and role of these evolutionary conserved and important chromosome proteins.