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Regulation of oocyte-specific chromatin organisation during prophase I by the histone demethylase Kdm5/Lid and other proteinsZhaunova, Liudmila January 2017 (has links)
In Drosophila oocytes, chromosomes undergo dynamic reorganisation during the prophase of the first meiotic division. This is essential to prepare chromatin for synapsis, recombination and consequent chromosome segregation. The progression of meiotic prophase I is well described, while the molecular mechanisms and regulation of these dramatic chromosomal reorganisations are not well understood. Histone modifying enzymes are major regulators of chromatin structure, however, our knowledge of their roles in meiotic prophase I is still limited. In this work, I investigated the role of the histone demethylase Kdm5/Lid, which removes one of the trimethyl groups at Lys4 of Histone 3 (H3K4me3). I showed that Kdm5/Lid is important for the assembly of the synaptonemal complex, pairing of homologous centromeres, and the karyosome formation. Additionally, Kdm5/Lid promotes crossing over and therefore ensures accurate chromosome segregation. Although loss of Kdm5/Lid dramatically increased the level of H3K4me3 in oocytes, catalytically inactive Kdm5/Lid rescued the above cytological defects. Thereby, I found that Kdm5/Lid regulates chromatin architecture in meiotic prophase I oocytes independently of its demethylase activity. To further identify the regulators of meiotic chromatin organisation during prophase I, I carried out a small-scale RNAi screen for karyosome defects. I found that depletion of ubiquitin ligase components, SkpA, Cul-3 and Ubc-6, disrupted the karyosome formation and the assembly of the synaptonemal complex. The success of the small-scale screen motivated me to initiate the genome-scale RNAi screen for karyosome defects. I found 40 new genes that, when depleted, strongly impaired karyosome morphology. Further studies are required to confirm and elucidate their role in chromatin organisation in oocytes. Overall, my findings have advanced our understanding of the regulation of chromatin reorganisation during oocyte development. Because of the conservation between Drosophila and human meiosis, this study provides novel insights into the regulation of meiotic progression in human oocytes.
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Replication, recombination and chromosome segregation in Escherichia coliWhite, Martin A. January 2010 (has links)
SbcCD has been shown to cleave a DNA hairpin formed by a palindromic DNA sequence on the lagging strand template of the E. coli chromosome. This activity was exploited to create a unique system for inducing a single site-specific DNA double-strand break (DSB) once per replication cycle. First, this work shows that the SOS response induced by this DSB is only essential for viability following multiple cycles of cleavage and repair. Next, the SOS-inducible inhibitor of cell division SfiA is shown to be dispensable for survival, despite demonstrating that cleavage of the palindrome causes both an increase in cell size and a delay in nucleoid segregation. A model of the E. coli cell cycle is presented to reconcile the observation that growth under chronic DSB induced conditions has no effect on generation time despite causing an increase in cell size. This system of DSB induction was then coupled with fluorescence markers on both sides of the palindrome to visualise the consequence of the DSB in vivo. Cleavage of the DNA hairpin by SbcCD in a recAmutant was used to selectively degrade the chromosome that replicated the palindrome on the lagging strand of replication, allowing two genetically identical sister chromosomes to be distinguished. This approach was used to show that chromosome segregation in E. coli is not random, but results in the segregation of lagging strand replicated DNA to mid-cell and leading strand replicated DNA to cell poles. Finally, this system for visualising the site of an inducible DSB was optimised for use in various other mutant backgrounds to allow the events of DSB repair to be dissected. This work provides a solid basis for further investigation into the relationship between replication, recombination and chromosome segregation in the model organism E. coli.
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Studies of chromosome structure and movement in C. elegans /Stear, Jeffrey Hamilton. January 2003 (has links)
Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 59-68).
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Cohesin proteins SMC1 and SMC3 : roles in aneuploidy and in meiotic chromosome dynamics /James, Rosalina Dee. January 2002 (has links)
Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 89-99).
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Investigation of Post-Translational Modification and Function of the Yeast Plasmid Partitioning Proteins Rep1 and Rep2Pinder, Jordan Benjamin 04 October 2011 (has links)
The 2-micron circle of Saccharomyces cerevisiae is one of a small number of similar
DNA plasmids found only in budding yeast. To understand how this cryptic parasite persists,
despite conferring no advantage to the host, I investigated the plasmid-encoded Rep1 and
Rep2 proteins. Interaction of Rep1 and Rep2 with each other and with the plasmid STB locus
is required for equal partitioning of plasmid copies at mitosis. The Rep proteins also repress
expression of Flp, the recombinase that mediates plasmid copy-number amplification. In this
study, absence of Rep1 and Rep2, or over-expression of the plasmid-encoded Raf antirepressor,
increased expression of a longer, novel FLP transcript. Translation of this mRNA
may explain elevated Flp activity at low plasmid copy number. Raf competed for Rep2 selfassociation
and interaction with Rep1, suggesting the mechanism of Raf anti-repression.
Deletion analysis identified a target site for Rep protein repression of FLP that is also
repeated in the STB locus, suggesting this as the sequence required for Rep protein
association with both regions of the plasmid.
Distinct roles for Rep1 and Rep2 were identified; Rep1 was found to depend on Rep2
for post-translational stability, with Rep2 dependent on Rep1 for stable association with STB.
Lysine-to-arginine substitutions in Rep1 and Rep2 impaired their association with the host
covalent-modifier protein SUMO, suggesting these were sites of sumoylation. The
substitutions did not affect interaction of the Rep proteins with each other or their stability
but did perturb plasmid inheritance, suggesting that Rep protein sumoylation contributes to
their plasmid partitioning function. When Rep1 was mutant, both Rep proteins lost their
normal localization to the nuclear foci where 2-micron plasmids cluster, and were impaired
for association with STB, supporting this as the cause of defective plasmid inheritance. The
potential sumoylation-dependent association of the Rep proteins with the 2-micron plasmid
partitioning locus suggests the plasmid has acquired a strategy common to eukaryotic viral
and host genomes that depend on sumoylation of their segregation proteins for faithful
inheritance. Collectively, my results shed light on how the 2-micron plasmid maintains the
delicate balance of persisting without harming its host.
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Analysis of SUMO dynamics and functions during meiosis in oocytes / 卵母細胞の減数分裂におけるSUMOの動態および機能の解析 / # ja-KanaDing, Yi 25 September 2018 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21400号 / 生博第401号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松崎 文雄, 教授 石川 冬木, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM
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Régulation dynamique de l’association des cohésines aux chromosomes, établissement et maintien de la cohésion des chromatides sœurs / Dynamic regulation of cohesin association with chromosomes, sister chromatid cohesion establishment and maintenanceFeytout, Amélie 09 December 2010 (has links)
Le complexe cohésine maintient associées les chromatides sœurs depuis la réplication jusqu’à leur ségrégation en mitose. Une question majeure est de comprendre comment la cohésion est établie lors de la phase S. Chez les mammifères et S. pombe, les cohésines sont associées de manière labile aux chromosomes pré-réplicatifs et l’établissement de la cohésion en phase S s’accompagne de la stabilisation de l’association des cohésines aux chromosomes. L’objectif de ce travail est de comprendre comment la dynamique des cohésines est régulée et comment son inhibition créée la cohésion.En G1 les cohésines associées aux chromosomes s’échangent avec le pool soluble et leur dissociation dépend de Pds5 et Wapl. La première partie de ce travail présente les résultats d’un crible génétique visant à identifier de nouveaux régulateurs de la dynamique des cohésines.L’établissement de la cohésion nécessite l’acétyltransférase Eso1 mais pas en contexte Δwpl1, indiquant que la seule mais essentielle fonction d’Eso1 est de s’opposer à celle de Wapl. L’acétylation de Smc3 par Eso1 contribue mais n’est pas suffisante pour contrecarrer Wapl, suggérant l’existence d’un autre événement dépendant d’Eso1. En G1, Pds5 agit avec Wapl pour dissocier les cohésines des chromosomes mais après la phase S, Pds5 est requise pour leur maintien sur les chromosomes et pour la cohésion à long terme. Pds5 co-localise avec la fraction stable de cohésines mais pas Wapl. Nous suggérons un modèle dans lequel la cohésion est créée par deux événements d’acétylation couplés à la progression de la fourche de réplication conduisant à l’éviction de Wapl des cohésines destinées à produire la cohésion. / Following DNA replication, sister chromatids are connected by cohesin to ensure their correct segregation during mitosis. How cohesion is created is still enigmatic. The cohesin subunit Smc3 becomes acetylated by ECO1, a conserved acetyl-transferase, and this change is required for cohesion. As in mammals, fission yeast cohesin is not stably bound to G1 chromosomes but a fraction becomes stable when cohesion is made. The aim of this work was to understand how cohesin dynamics is regulated and how the change in cohesin dynamics creates cohesion.In G1 chromatin bound cohesin exchange with the soluble pool and the unloading reaction relies in part on Wapl. The first part of this study reports on the identification of G1/S factors as new candidate regulators of cohesin dynamics.Following S phase a stable cohesin fraction is made. The acetyl-transferase Eso1 is not required for this reaction when the wpl1 gene is deleted. Yet, it is in wild-type cells, showing that the sole but essential Eso1 function is counteracting Wapl. Eso1 acetylates the cohesin sub-unit Smc3. This renders cohesin less sensitive to Wapl but does not confer the stable binding mode, suggesting the existence of a second Eso1-dependent event. The cohesin sub-unit Pds5 act together with Wapl to promote cohesin removal from G1 chromosomes but after S phase Pds5 is essential for cohesin retention on chromosomes and long term cohesion. Pds5 co-localizes with the stable cohesin fraction whereas Wapl does not. We suggest a model in which cohesion establishment is made by two acetylation events coupled to fork progression leading to Wapl eviction while keeping Pds5 on cohesin complexes intended to make cohesion.
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Structure-function studies of the bacterial dsDNA translocase FtsKGraham, James Edward January 2010 (has links)
DNA translocases are molecular motors that use energy from nucleotide triphosphate (NTP) hydrolysis to move along, pump, remodel or clear DNA. Unlike helicases, double-stranded DNA (dsDNA) translocases do not unwind DNA; their action has no net product apart from inducing supercoils as a result of groove-tracking, which has hampered their characterisation. Many dsDNA translocases appear to have biased directionality. However, the inherent symmetry of dsDNA requires that translocase activity is regulated by specific sequences or through modulation by interaction partners. FtsK is a highly conserved bacterial cell-division protein, localised to the dividing septum, that coordinates chromosome segregation with cytokinesis. It is responsible for the resolution of chromosome dimers by activating the tyrosine recombinases XerCD bound to the 28bp chromosomal site dif. The C-terminal domain of FtsK (FtsKC) is a dsDNA translocase (speed ~5 kb/s, stall force ~60 pN) most closely related to superfamily 4 helicases and is active as a hexameric ring. A winged-helix subdomain at the C-terminus of FtsKC, FtsKgamma, binds to specific 8 bp sequences, KOPS, that are polarised in the bacterial chromosome from the origin to towards dif. FtsKgamma also interacts with XerD, activating it for catalysis. Studies of FtsK translocation have differed over whether KOPS act as a loading or a reversal sequence for FtsK. In Chapter 2, I use a continuous ensemble assay for dsDNA translocation to show that FtsK initiates rapidly at KOPS, with loading dependent on FtsKgamma. Translocation requires moderately cooperative ATP binding, while ATP hydrolysis has a more relaxed cooperativity. I have determined the ATP coupling efficiency of translocation to be ~1.6 bp/ATP, in line with theoretical estimates. Though FtsK probably strips most proteins from DNA, I show in Chapter 3 that FtsK stops translocating when it encounters XerCD bound to dif. The interaction is most likely a specific down-regulation, but surprisingly does not depend on FtsKgamma or on the catalytic or synaptic activity of XerCD. In Chapter 4, I show some preliminary structural data of FtsKC bound to dsDNA, with the aim of determining the first high resolution structure of a ring dsDNA translocase bound to nucleic acid.
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How and why to stop and wait : a graduate education in mechanisms and benefits of suspended animation /Goldmark, Jesse P. January 2006 (has links)
Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 54-58).
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Characterization of the budding yeast centromeric histone H3 variant, Cse4 /Collins, Kimberly A. January 2006 (has links)
Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 104-113).
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