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

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

Genetic analysis of the Replication Protein A large subunit family in Arabidopsis reveals unique and overlapping roles in DNA repair, meiosis and DNA replication

Replication Protein A (RPA) is a heterotrimeric protein complex that binds single-stranded DNA. In plants, multiple genes encode the three RPA subunits (RPA1, RPA2 and RPA3), including five RPA1-like genes in Arabidopsis. Phylogenetic analysis suggests two distinct groups composed of RPA1A, RPA1C, RPA1E (ACE group) and RPA1B, RPA1D (BD group). ACE-group members are transcriptionally induced by ionizing radiation, while BD-group members show higher basal transcription and are not induced by ionizing radiation. Analysis of rpa1 T-DNA insertion mutants demonstrates that although each mutant line is likely null, all mutant lines are viable and display normal vegetative growth. The rpa1c and rpa1e single mutants however display hypersensitivity to ionizing radiation, and combination of rpa1c and rpa1e results in additive hypersensitivity to a variety of DNA damaging agents. Combination of the partially sterile rpa1a with rpa1c results in complete sterility, incomplete synapsis and meiotic chromosome fragmentation, suggesting an early role for RPA1C in promoting homologous recombination. Combination of either rpa1c and/or rpa1e with atr revealed additive hypersensitivity phenotypes consistent with each functioning in unique repair pathways. In contrast, rpa1b rpa1d double mutant plants display slow growth and developmental defects under non-damaging conditions. We show these defects in the rpa1b rpa1d mutant are likely the result of defective DNA replication leading to reduction in cell division

Negative Cell Cycle Regulation and DNA Damage-inducible Phosphorylation of the BRCT Protein 53BP1

In a screen designed to discover suppressors of mitotic catastrophe, we identified the Xenopus ortholog of 53BP1 (X53BP1), a BRCT protein previously identified in humans through its ability to bind the p53 tumor suppressor. X53BP1 transcripts are highly expressed in ovaries, and the protein interacts with Xp53 throughout the cell cycle in embryonic extracts. However, no interaction between X53BP1 and Xp53 can be detected in somatic cells, suggesting that the association between the two proteins may be developmentally regulated. X53BP1 is modified via phosphorylation in a DNA damage-dependent manner that correlates with the dispersal of X53BP1 into multiple foci throughout the nucleus in somatic cells. Thus, X53BP1 can be classified as a novel participant in the DNA damage response pathway. We demonstrate that X53BP1 and its human ortholog can serve as good substrates in vitro as well as in vivo for the ATM kinase. Collectively, our results reveal that 53BP1 plays an important role in the checkpoint response to DNA damage, possibly in collaboration with ATM

Transcription during meiosis in the fission yeast Schizosaccharomyces pombe

The meiotic cell cycle is the process by which diploid organisms divide to produce haploid gametes and consists of one round of DNA replication followed by two successive nuclear divisions. In the fission yeast, Schizosaccharomyces pombe, meiosis is initiated from G1 phase and involves a complex series of cellular events that lead to the production of four haploid ascospores. The periodic regulation of gene expression is an important mechanism of control of both mitotic- and meiotic-cell-cycle progression. During the mitotic cell cycle of fission yeast a number of genes, including cdc22+, cdc18+ and cdt1+, are expressed specifically at the G1-S phase boundary. These genes are known to be under the control of MCB (MluI cell-cycle box) upstream-activating-sequence motifs and the MBF (MCB binding factor; also known as DSC1) complex. Here we show that control of gene expression during pre-meiotic G1-S-phase is mediated by an MBF-related transcription-factor complex acting upon similar MCB promoter motifs. Several genes, including rec11+, rec11+, cdc18+, and cdc22+, which contain MCB motifs in their promoter regions, are shown to be coordinately regulated during pre-meiotic S-phase. These genes can be divided into 'mitotic and meiotic' and 'meiotic specific' groups, which contain related but distinct arrangements of MCB motifs. MCB motifs are shown to be physiologically relevant during the meiotic cell cycle and to confer meiotic-specific transcription to a heterologous reporter gene. An MBF-like transcription factor complex that binds to MCB motifs is identified in meiotic cells. The effect of mutating and over-expressing individual components of MBF (cdc10+, res1+, res2+, rep1+ and rep2+) on cdc22+, rec8+ and ree11+ transcription during meiosis was examined. We found that cdc10+, res2+ and rep1+ are required for meiotic transcription, while rest has no role in this process. Surprisingly, manipulation of the mitotic-specific rep2+ gene had an effect on 'meiotic specific' but not 'mitotic and meiotic' MCB-regulated transcription during the meiotic cell cycle. This indicates that Rep2p might have a role in allowing the MBF complex to distinguish between 'mitotic and meiotic' and 'meiotic specific' MCB motifs. This work is the first demonstration in yeast of a role for MCB motifs in control of transcription during meiosis, and identifies a meiotic MBF-like transcription-factor complex

Analysis of the transcriptional program governing meiosis and gametogenesis in yeast and mammals

During meiosis a competent diploid cell replicates its DNA once and then undergoes two consecutive divisions followed by haploid gamete differentiation. Important aspects of meiotic development that distinguish it from mitotic growth include a highly increased rate of recombination, formation of the synaptonemal complex that aligns the homologous chromosomes, as well as separation of the homologues and sister chromatids during meiosis I and II without an intervening S-phase. Budding yeast is an excellent model organism to study meiosis and gametogenesis and accordingly, to date it belongs to the best studied eukaryotic systems in this context. Knowledge coming from these studies has provided important insights into meiotic development in higher eukaryotes. This was possible because sporulation in yeast and spermatogenesis in higher eukaryotes are analogous developmental pathways that involve conserved genes. For budding yeast a huge amount of data from numerous genome-scale studies on gene expression and deletion phenotypes of meiotic development and sporulation are available. In contrast, mammalian gametogenesis has not been studied on a large-scale until recently. It was unclear if an expression profiling study using germ cells and testicular somatic control cells that underwent lengthy purification procedures would yield interpretable results. We have therefore carried out a pioneering expression profiling study of male germ cells from Rattus norvegicus using Affymetrix U34A and B GeneChips. This work resulted in the first comprehensive large-scale expression profiling analysis of mammalian male germ cells undergoing mitotic growth, meiosis and gametogenesis. We have identified 1268 differentially expressed genes in germ cells at different developmental stages, which were organized into four distinct expression clusters that reflect somatic, mitotic, meiotic and post-meiotic cell types. This included 293 yet uncharacterized transcripts whose expression pattern suggests that they are involved in spermatogenesis and fertility. A group of 121 transcripts were only expressed in meiotic (spermatocytes) and postmeiotic germ cells (round spermatids) but not in dividing germ cells (spermatogonia), Sertoli cells or two somatic control tissues (brain and skeletal muscle). Functional analysis reveals that most of the known genes in this group fulfill essential functions during meiosis, spermiogenesis (the process of sperm maturation) and fertility. Therefore it is highly possible that some of the �30 uncharacterized transcripts in this group also contribute to these processes. A web-accessible database (called reXbase, which was later on integrated into GermOnline) has been developed for our expression profiling study of mammalian male meiosis, which summarizes annotation information and shows a graphical display of expression profiles of every gene covered in our study. In the budding yeast Saccharomyces cerevisiae entry into meiosis and subsequent progression through sporulation and gametogenesis are driven by a highly regulated transcriptional program activated by signal pathways responding to nutritional and cell-type cues. Abf1p, which is a general transcription factor, has previously been demonstrated to participate in the induction of numerous mitotic as well as early and middle meiotic genes. In the current study we have addressed the question how Abf1p transcriptionally coordinates mitotic growth and meiotic development on a genome-wide level. Because ABF1 is an essential gene we used the temperature-sensitive allele abf1-1. A phenotypical analysis of mutant cells revealed that ABF1 plays an important role in cell separation during mitosis, meiotic development, and spore formation. In order to identify genes whose expression depends on Abf1p in growing and sporulating cells we have performed expression profiling experiments using Affymetrix S98 GeneChips comparing wild-type and abf1-1 mutant cells at both permissive and restrictive temperature. We have identified 504 genes whose normal expression depends on functional ABF1. By combining the expression profiling data with data from genome-wide DNA binding assays (ChIPCHIP) and in silico predictions of potential Abf1p-binding sites in the yeast genome, we were able to define direct target genes. Expression of these genes decreases in the absence of functional ABF1 and whose promotors are bound by Abf1p and/or contain a predicted binding site. Among 352 such bona fide direct target genes we found many involved in ribosome biogenesis, translation, vegetative growth and meiotic developement and therefore could account for the observed growth and sporulation defects of abf1-1 mutant cells. Furthermore, the fact that two members of the septin family (CDC3 and CDC10 ) were found to be direct target genes suggests a novel role for Abf1p in cytokinesis. This was further substantiated by the observation that chitin localization and septin ring formation are perturbed in abf1-1 mutant cells

ATM-Mediated Transcriptional and Developmental Responses to γ-rays in Arabidopsis

ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of γ-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR. The transcription burst was almost exclusively AtATM-dependent or weakly AtATR-dependent, and followed two major trends of expression in atm: (i)-loss or severe attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one to distinguish IR/ATM pathway constituents. Our data provide a large resource for studies on the interaction between plant checkpoints of the cell cycle, development, hormone response, and DNA repair functions, because IR-induced transcriptional changes partially overlap with the response to environmental stress. Putative connections of ATM to stem cell maintenance pathways after IR are also discussed

Arabidopsis ULTRAVIOLET-B-INSENSITIVE4 maintains cell division activity by temporal inhibition of the anaphase-promoting complex/cyclosome

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that regulates progression through the cell cycle by marking key cell division proteins for destruction. To ensure correct cell cycle progression, accurate timing of APC/C activity is important, which is obtained through its association with both activating and inhibitory subunits. However, although the APC/C is highly conserved among eukaryotes, no APC/C inhibitors are known in plants. Recently, we have identified ULTRAVIOLET-B-INSENSITIVE4 (UVI4) as a plant-specific component of the APC/C. Here, we demonstrate that UVI4 uses conserved APC/C interaction motifs to counteract the activity of the CELL CYCLE SWITCH52 A1 (CCS52A1) activator subunit, inhibiting the turnover of the A-type cyclin CYCA2;3. UVI4 is expressed in an S phase-dependent fashion, likely through the action of E2F transcription factors. Correspondingly, uvi4 mutant plants failed to accumulate CYCA2; 3 during the S phase and prematurely exited the cell cycle, triggering the onset of the endocycle. We conclude that UVI4 regulates the temporal inactivation of APC/C during DNA replication, allowing CYCA2;3 to accumulate above the level required for entering mitosis, and thereby regulates the meristem size and plant growth rate

Analysis of chromosomal rearrangements after replication restart

Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCRs) and copy-number variations (CNVs). GCRs and CNVs underlie human genomic disorders and are a feature of cancer. During cancer development, environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs and CNVs are proposed to contribute to cancer development and therapy resistance. Using an inducible system that arrests replication forks at a specific locus in fission yeast, chromosomal rearrangement was investigated. In this system, replication restart requires homologous recombination. However, it occurs at the expense of gross chromosomal rearrangements that occur by either faulty template usage at restart or after the correctly restarted fork U-turns at inverted repeats. Both these mechanisms of chromosomal rearrangement generate acentric and reciprocal dicentric chromosomes. The work in this thesis analyses the timing of replication restart and appearance of chromosomal rearrangements in a single cell cycle after induction of fork stalling. This research also identifies the recombination-dependent intermediates corresponding to the two pathways of rearrangements. Moreover, the DNA integrity checkpoint responses after replication fork arrest, homologous recombination dependent replication restart, and the accumulation of GCRs are investigated