S. coelicolor uses an active chromosome partitioning system for developmentally-regulated genome segregation, which is associated with spore formation. There are four known trans-acting segregation proteins (ParA, ParB, ParJ and Scy) and cis-acting centromere-like sites (parS). parA encodes a Walker-type ATPase that is required for efficient DNA segregation and proper placement of the ParB-parS nucleoprotein complexes. A paralogue of ParA is encoded by the S. coelicolor genome, SCO1772 (named ParH), that has 45% identical residues to ParA. In S. coelicolor aerial hyphae, a ∆parH mutant produces 5% of anucleate spores. In this study, ParH was identified as a novel interaction partner of S. coelicolor ParB. However, a Walker A motif K99E substitution in ParH and removal an N-terminal extension in ParH impaired interaction between ParH and ParB, as judged by bacterial two-hybrid analyses. ParH-EGFP localization resembles the evenly-spaced localization pattern of ParH-EGFP in aerial hyphae, which might suggest that ParH colocalizes with ParB. A parH-null mutant appears to be unable to properly organize the oriC regions within a subset of prespores, as judged by ParB-EGFP foci. In this study, through a random chromosomal library screening, a novel protein that interacts with ParA and ParH was also identified. HaaA (ParH and ParA Associated protein A) is required for proper chromosome segregation and is one of the 24 signature proteins of the Actinomycetes that are not found in other bacterial lineages. A bacterial two-hybrid analysis showed that HaaA interacts with itself and interaction between ParH and ParA was through the C-terminal unstructured region. Interaction between HaaA and ParA and ParA-like proteins was conserved in other Actinomycetes, such as S. venezuelae, C. glutamicum and M. smegmatis. There was no evidence for interaction with other tested segregation proteins. In addition, a haaA insertion-deletion mutant strain revealed that loss of HaaA affected chromosome segregation (6% anucleate spores) and HaaA-EGFP localizes within spores of the mature spore chains. Together these data revealed new information to further understand chromosome segregation in S. coelicolor. / Bayer School of Natural and Environmental Sciences; / Biological Sciences / PhD; / Dissertation;

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Genetics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 34-38).

No description available.

Meiosis is a specialized form of cell division where homologous chromosomes are segregated in meiosis I before sister chromatids are segregated in meiosis II. To establish this pattern, a number of changes to the mitotic chromosome segregation machinery are put in place. Firstly, sister kinetochores orient towards the same pole in meiosis I (mono-orientation). Secondly, homologue recombination creates chiasmata, which link homologues together. And thirdly, cohesin, the molecule that holds sister chromatids together, is cleaved in a step-wise manner. This is achieved because the Shugoshin (Sgo1) protein recruits protein phosphatase 2A (PP2A) to centromeres to counteract cohesin phosphorylation, which is required for its cleavage. The work presented here has investigated two critical aspects of cohesin protection: firstly, how cohesin protection is deactivated in meiosis II and, secondly, how a meiosis-specific protein called Spo13 helps to set up cohesin protection in meiosis I. Previously, our lab had shown that Sgo1 is removed from chromosomes when sister chromatids come under tension during mitosis. I therefore sought to investigate whether sister kinetochore mono-orientation allows Sgo1 to stay on centromeres during meiosis I and carry out its protective function. To this end, I modified meiosis I chromosomes to lack both chiasmata and mono-oriented kinetochores. Under these conditions, where sister chromatids are forced to be under tension in metaphase I, Sgo1 is undetectable on chromosomes. As a consequence, centromeric cohesin is largely lost in anaphase I leading to the premature separation of sister chromatids in a fraction of cells. Since mono-orientation of sister kinetochores is exclusive to meiosis I, these findings suggest that Sgo1 localisation is influenced by sister kinetochore tension in both mitosis and meiosis. Therefore, our findings suggest a mechanism that could contribute to the deprotection of cohesin in meiosis II. However, loss of cohesin protection upon bi-orientation is not complete, suggesting that other factors are involved in the efficient protection and deprotection of cohesin. One such factor is the meiosis-specific protein Spo13, which had previously been shown to be required for cohesin protection as well as kinetochore monoorientation. Although it had been suggested that Spo13 regulates Sgo1 recruitment to centromeres, I could not find any evidence to support a loss of Sgo1, or PP2A, in spo13Δ cells. Additionally, even when Sgo1 is stabilised and clearly visible in anaphase I of spo13Δ mutants, pericentromeric cohesion is still defective. Therefore, I investigated the effect that polo kinase Cdc5, an interactor of Spo13, has on Sgo1. While cellular Sgo1 levels are increased in response to Cdc5 loss, this effect seems to be independent of Spo13. However, Spo13 is required for proper levels of Cdc5 at centromeres and the centromeric recruitment of Cdc5 by Spo13 is likely to be functionally important because tethering of Cdc5 to kinetochores rescued the mono-orientation phenotype of spo13Δ cells. In contrast, I found no evidence that the Spo13-Cdc5 interaction is required for cohesin protection. Meiotic overexpression of SPO13 enhances cohesin protection in meiosis I, apparently independent of its robust interaction with Cdc5, and causes increased Sgo1 enrichment at centromeres. This suggested that Spo13 might recruit Sgo1 to cohesin itself to facilitate its protection. Although I could not detect a loss of Sgo1-cohesin interaction in spo13Δ cells, tethering of Sgo1 to cohesin restores pericentromeric Rec8 to spo13Δ mutants in anaphase I. Surprisingly, sister chromatids still segregate in this case, suggesting that pericentromeric cohesion is defective, despite maintenance of Rec8. Furthermore, inhibition of either one of the cohesin kinases, DDK and Hrr25, restores sister chromatid cohesion to spo13Δ cells. Therefore, the findings in this study suggest that Spo13 is at the centre of a complex regulatory network that coordinates cohesin protection and sister chromatid cohesion in meiosis I.

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references. Also available online through Digital Dissertations.

Centromere identity is integral for proper kinetochore formation and chromosome segregation. In most species chromosomes have a centromere at a defined locus that is propagated across generations. The histone H3 variant CENP-A acts as an epigenetic mark for centromere identity in most species studied. CENP-A is absent from the inactivated centromere on dicentric chromosomes and present at neocentromeres that form on non-centromeric sequences. Thus, the canonical centromere sequence is neither necessary nor sufficient for centromere function. Nevertheless, centromeres are generally associated with particular sequences. Understanding the organisation of centromeric sequence features will provide insight into centromere function and identity. In this study I use the fission yeast Schizosaccharomyces pombe model system to address the relationship between CENP-ACnp1 and centromeric sequence features. These analyses reveal that CENP-ACnp1 nucleosomes are highly positioned within the central domain by large asymmetric AT-rich gaps. The same sequence features underlying CENP-ACnp1 positioning are conserved in the related species S. octosporus, but are not found at neocentromeres, suggesting that they are important but non-essential for centromere function. CENP-ACnp1 over-expression leads to ectopic CENP-ACnp1 incorporation primarily at sites associated with heterochromatin, including the sites where stable neocentromeres form. Ectopic CENP-ACnp1 also occupies additional sites within the central domain that are not occupied in cells with wild-type CENP-ACnp1 levels. In wild-type cells CENP-ACnp1 occupied sites are likely also occupied by H3 nucleosomes or the CENP-T/W/S/X nucleosome-like complex in a mixed population. Several candidate proteins were investigated to determine a protein residing in the large gaps between CENP-ACnp1 nucleosomes could be identified. No proteins could be localised to the AT-rich gaps between CENP-ACnp1 nucleosomes, but the origin recognition complex in a promising candidate. The results presented in this thesis demonstrate that nucleosomes within the fission yeast centromere central domain are highly positioned by sequence features in a conserved manner. This positioning also allows for another complex, possibly the origin recognition complex, to bind to DNA. Nucleosome positioning, DNA replication, and transcription could individually and collectively influence CENP-ACnp1 assembly and centromere function. Further experiments in fission yeast will continue to provide insight into the general properties of centromere function and identity.

The degree of DNA supercoiling in the cell is carefully controlled by DNA topoisomerases. These enzymes catalyze the passage of individual DNA strands (Type I DNA topoisomerases), or double helices (Type II DNA topoisomerases) through one another. The purpose of the present study is to conduct a detailed analysis of the topo llα and β mRNAs expressed in several vertebrate cell lines. The final aim of this project is to analyze the relative roles of topo llα in chromatin condensation and chromosome segregation during mitosis, by performing topo llα gene targeting experiments in the DT 40 chicken lymphoblastoid cell line. The knock-out strategy was based on the observation of a high rate of homologous recombination versus random integration in the DT40 cell line. The topo llα gene was shown to be located on the chicken chromosome 2 (APM unpublished), for which the DT40 cell line is trisomic. The targeting vector completely replaced the 32 kb topo IIα genomic locus, generating a topo llα (-/+/+)cell line, which showed an increased resistance to topo II inhibitors. Paradoxically, 150 uM etoposide or 100 uM mitoxanthrone induced apoptosis within 5 hours in the topo llα (-1+1+) cell line, more rapidly as compared to the normal DT 40 cells. A topo IIα (-I-I+) cell line has also been generated. This study revealed the presence of evolutionarily conserved alternatively spliced forms of both topo llα and β isoforms between birds and humans. Hybridization screening of two chicken cDNA libraries, MSB-1 and DU249, revealed the presence of two distinct forms of both topo llα and β cDNAs. One form of topo llα, designated topo llα-1, encodes the chicken topo llα amino acid sequence previously reported (dbjiAB007445) in the database (unpublished). The second form, designated topo llα-2, encodes a protein containing an additional 35 amino acids inserted after Lysine-322 of chicken topo IIα-1 protein sequence. In the case of topo 11(3 mANA, one form, designated topo IIβ-1, encodes the protein already described (dbjiAB007446). The second form, tapa IIβ-2, would encode a protein missing the next 86 amino acids after Valine-25 in tapa II β-1 protein sequence. The tapa 11β variant is positioned similarly to one previously described in human (Hela) cells. The 5 amino acid insertion in the human tapa 11β-2 variant follows v23. In chicken cells, a longer insertion of 86 amino acids sequence follows v25, the homologous position in the tapa 11β protein. In human cells, the situation with tapa llα is more complex, as revealed by RT-PCR experiments (APM, unpublished) which generated several bands. One of these amplified species was found to contain a 36 amino acids insertion, positioned after residue K321 in the human tapa IIα cDNA, similarly to chicken tapa IIα-2 variant. The second human tapa llα spliced form cDNA was shown to contain a 26 amino acids insertion after residue A401 in the canonical human tapa llα protein sequence. The third cDNA variant isolated from human cells was described to encode a 81 amino acids insertion after residue Q355 positioned within the known human tapa IIα protein. It appears possible that the posttranslational modifications of the a-2 and β-2 isoforms may differ substantially from those of the canonical a-1 and β-1 isoforms. Such variant proteins could fulfil specialized functions, which might be tissue or cell-type specific. In summary, two novel forms of tapa llα and β cDNAs have been identified in three chicken cell lines. These spliced versions of both tapa llα and 13 isoforms seem to be evolutionary conserved, with similar forms occurring in their human counterparts. Future functional analysis of vertebrate tapa IIα and β will have to account for the existence of these novel isoforms, which might encode proteins that may exhibit different regulation of their subcellular localization, interaction with other proteins, or catalytic activity.

Sister chromatid cohesion is essential for accurate chromosome segregation. Cohesion is generated by cohesin, a conserved multi-subunit protein complex composed of four core subunits: Smc1, Smc3, Scc1, and Scc3. Cohesin holds sister chromatids together in mitotic cells starting from S-phase, when DNA replicates, until their separation at the onset of anaphase where its Scc1 subunit is cleaved. In budding yeast, most Scc1 is destroyed by cleavage at anaphase and is only re-synthesised in late G1, whereupon it associates with the unreplicated chromatin. Although sister chromatid cohesion is known to be mediated by a topological interaction of cohesin complexes around sister DNAs, the nature of cohesin`s interaction with chromatin before DNA replication remains to be elucidated. My project aims to develop a new system in order to find out whether ‘non-cohesive’ cohesin interacts with chromatin topologically. This is important for two main reasons. Firstly, understanding the physical nature of cohesin’s interaction with chromatin before DNA replication is essential for determining how cohesion is established during DNA replication. Another reason is that most cohesin in multicellular organisms is associated with the unreplicated chromatin of post mitotic cells where it regulates transcription. How cohesin mediates gene expression is unknown. Understanding how cohesin binds unreplicated chromatin may therefore bring insights into the mechanisms by which cohesin complex performs its non-canonical functions. In order to address this, we needed a situation where cohesin is already loaded onto chromosomes, but either DNA replication or cohesion establishment is prevented. Therefore, we used a temperature sensitive allele of Eco1 (required for establishment of cohesion). Quantitative measurement of cohesin levels on chromosomes in either wild type allele or temperature sensitive allele of Eco1 showed that the amount of cohesin associated with centromeric and inner pericentromeric regions in both strains are almost indistinguishable from each other throughout the whole cell cycle. Despite normal levels of cohesin, we confirmed by minichromosomal assay that no sister chromatid cohesion is established in the absence of functional Eco1 protein. If “non-cohesive” cohesin interacts with the chromatin in a topological manner when there is no sister chromatid cohesion, then its association with chromatin should be resistant to denaturing conditions in the presence of a modified version of the cohesin complex that can be covalently circularized. To test this prediction, a cross-linkable cohesin molecule was needed, which should be resistant to SDS denaturation and should not have major cohesion defects due to the modifications making it to be cross-linkable. The previously created cross-linkable cohesin molecule had cohesion defects due to the presence of Smc3-Scc1 fusion protein. In addition, this fusion alone could bypass the requirement for Eco1, and therefore we could not test how “non-cohesive” cohesin interacts with chromatin, using this version of cross-linkable cohesin complex. We tried two different methods to conditionally close Smc3/Scc1 interface in a way resistant to protein-denaturants and create a new cross-linkable cohesin complex. In our first attempt, the C-terminus of Smc3 and the N-terminus of Scc1 were fused to FRB and FKBP12 respectively, proteins that can form a complex upon addition of rapamycin. Crystal structure of the ternary complex of FKP12/rapamycin/FRB enabled us to design cysteine pairs for the crosslinking of FRB and FKBP12 only in the presence of rapamycin. A more efficient in vivo crosslinking was achieved between the Smc3 and Scc1 in our second attempt. Amino acids within the coiled coil region of Smc3 were replaced by the unnatural photo-cross-linkable amino acid ρ-benzoyl-phenylalanine that can be induced to form covalent bonds with neighbouring proteins (T.Gligoris, unpublished data). Photo and chemically cross-linkable interfaces of cohesin were then integrated with each other to generate a new version of cross-linkable cohesin molecule.