In the fission yeast, Schizosacchromyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both stabilize stalled replication forks and to prevent premature entry into mitosis. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, my study shows that mutants defective in DNA replication initiation require the Chk1 kinase, rather than Cds1. This suggests that failed initiation events can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. I refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). The data shows that Chk1 is active in rid mutants when grown under semi-permissive conditions, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, an adaptor protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation. Like Mrc1, Swi1 and Swi3 have been hypothesized as a part of the replication fork protection complex (RFPC). They are required for maintaining the viability of rid mutants, but are not essential for activation of Chk1 in response to failed initiation events. This suggests that Mrc1 in conjunction with Swi1 and Swi3 function in a similar pathway to alleviate replicative stress resulting from defects in DNA replication initiation. Using flow cytometry, I demonstrate that inhibition of DNA replication initiation has no significant impact on the duration of S phase, suggesting dormant origins might be activated in response to defects in DNA replication initiation. Fission yeast Rad22 is implicated in forming nuclear foci in response to damaged DNA. By tracking YFP-labeled Rad22, I screened for potential DNA damage in rid mutants grown at semi-permissive temperatures, and the results show that DNA damage occurs as the result of defects in DNA replication initiation. I also identified camptothecin, a DNA topoisomerase I inhibitor that can at low dose (2 µM) induce the rid phenotype, suggesting our assay (Chk1-dependent, Cds1-independent) can be used to screen small molecule inhibitors that interfere with the initiation step of DNA replication.

No description available.

p13^suc1 from schizosaccharomyces pombe is a member of a family of non-enzymatic cell cycle regulatory proteins called CKS for <strong>C</strong>yclin-<strong>D</strong>ependent kinases <strong>S</strong>ubunit. Other members of this family include CKS1 (S. cerevisiae</em?), CksHsl and CksHs2 (Homo sapiens). The CDKs (CDK1-CDK8) for <strong>C</strong>yclin-<strong>D</strong>ependent <strong>K</strong>inases are a class of Ser/Thr kinases that regulate the cell cycle. The suc1⁺ gene was initially identified as a seppresor of certain CDKl temperature sensitive mutations. Despite the wealth of crystallographic models available plus ample - albeit, sometimes conflicting - evidence from genetics and biochemical studies, an account of the exact physiological role of the CKS proteins remains an elusive goal. In a quest to identify the residues and hence the particular surface region involved in mediating protein-protein interactions with CDK2,1 employed the X-ray structure of Suc1 at 2.7A resolution as guide for a site-specific mutagenesis study. Comparative biochemical and biophysical characterisation of Suc1 and the mutants led to the conclusion that isoleucine-94 and Leucine-96 (located in the hydrophobic patch) are involved in mediating protein-protein interactions with GST-CDK2. This conclusion has since been confirmed by the publication of the X-ray structure of monomeric CksHs1l in a complex with CDK2 by Bourne et al., 1996 (Cell 84: 863-874). An extension of the mutational study to test the hypothesis that Suc1 may utilise conserved residues at the anion-binding site to mediate protein-protein interactions with the Anaphase Promoting Complex (APC) is described. X-ray data has been collected on wild type Suc1 crystals at 100K to 2.3Å resolution. The structure has been resolved and refined to a crystallographic R-factor of 22.6%. S. pombe Suc1 exists as a zinc-stabilised, non strand-exchanged dimer in both the 2.1Å and 2.3Å models. Structural analyses of two Suc1 folding mutants are also presented. The cyclins (A - H) are positive regulatory subunits of CDKs. They share a high degree of homology over a region of about 100 amino acid residues called the "cyclin box". The determination of the crystal structure of cyclin A3 (an N-terminal truncated version of bovine cyclin A) and a CDK2-cyclin A3 complex by other workers has revealed the mechanism of activation of CDKs by cyclins. In S. pombe, the CDKl-cyclin B heterodimeric complex constitutes the mitotic kinase. In order to understand the specificity underlying the CDK-cyclin interaction, I embarked on a structural study of S. pombe cyclin B and CDK1. Both full- length proteins have proven intractable to attempts to overproduce them in a soluble form in E. coli for crystallisation studies. A truncated version of cyclin B (similar to cyclin A3) was designed, cloned and overproduced in E. coli. The cyclin B3 protein was directed into inclusion bodies as insoluble aggregates. Extensive attempts - both in vivo and in vitro - to produce a soluble cyclin B3 proved unsuccessful. Finally, an E. coli co-expression system designed to overproduce CDK1-cyclin A3, CDK1-cyclin B3, CDK1-cyclin B and CDK1-Suc1 complexes is described.

Stuttgart, Univ., Diss., 2005.

Thesis (Ph.D. in Biochemistry) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 93-103). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Techn. Hochsch., Diss., 2003--Aachen.

Techn. Universiẗat, Diss., 2005--Dresden.

In most eukaryotes methylation of histone H3 on lysine 9 (H3K9me) is the key post-translational modification required for the assembly of constitutive heterochromatin at centromeres and other chromosomal regions. H3K9me is bound by the chromodomain proteins HP1/Swi6 and the Suv39/Clr4 H3K9 methyltransferase itself suggesting that, once established, H3K9me might act as an epigenetic mark that can transmit the chromatin state independently of the initiator signal. However, it has not been demonstrated that H3K9me does indeed act as an epigenetic mark. Fission yeast represents an excellent system to address this question since S. pombe lacks DNA methylation and H3K9me is catalysed by the unique, non-essential H3K9 methyltransferase Clr4. To determine whether H3K9me carries epigenetic properties it is important to uncouple H3K9me from genomic domains that have the intrinsic ability to recruit the heterochromatin machinery. One way to solve this problem is to isolate H3K9me from its original context and investigate whether at an ectopic site H3K9me can self-propagate through cell division. To accomplish this, we tethered regulatable TetR-Clr4 fusion protein at euchromatic loci in fission yeast. This resulted in the assembly of an extensive domain of H3K9me-dependent heterochromatin that is rapidly disassembled following TetR-Clr4 release. Strikingly, the inactivation of Epe1, a putative histone demethylase, is sufficient to maintain the silent H3K9me-dependent heterochromatin at the tethering sites through mitotic and meiotic cell divisions in absence of TetR-Clr4. These results indicate that H3K9me acts as an epigenetic mark to maintain heterochromatin domains; however, a regulatory mechanism dependent on Epe1 exists to actively remove H3K9me and thus prevent heterochromatin from being transmitted when assembled at inappropriate regions of the genome.

The biogenesis of spliceosomal UsnRNPs is a highly elaborate cellular process that occurs both in the nucleus and the cytoplasm. A major part of the process is the assembly of the Sm-core particle, which consists of a ring shaped heptameric unit of seven Sm proteins (SmD1•D2•F•E•G•D3•B) wrapped around a single stranded RNA motif (termed Sm-site) of spliceosomal UsnRNAs. This process occurs mainly in the cytoplasm by the sequential action of two biogenesis factors united in PRMT5- and SMN-complexes, respectively. The PRMT5-complex composed of the three proteins PRMT5, WD45 and pICln is responsible for the symmetric dimethylation of designated arginine residues in the C-terminal tails of some Sm proteins. The action of the PRMT5- complex results in the formation of assembly incompetent Sm-protein intermediates sequestered by the assembly chaperone pICln (SmD1•D2•F•E•G•pICln and pICln•D3•B). Due to the action of pICln, the Sm proteins in these complexes fail to interact with UsnRNAs to form the mature Sm-core. This kinetic trap is relieved by the action of the SMN-complex, which removes the pICln subunit and facilitates the binding of the Sm-core intermediates to the UsnRNA, thus forming the mature Sm-core particle. The human SMN complex consists of 9 subunits termed SMN, Gemin2-8 and Unrip. So far, there are no available atomic structures of the whole SMN-complex, but structures of isolated domains and subunits of the complex have been reported by several laboratories in the past years. The lack of structural information about the entire SMN complex most likely lies in the biophysical properties of the SMN complex, which possesses an oligomeric SMN core, and many unstructured and flexible regions. These were the biggest roadblocks for its structural elucidation using traditional methods such as X-ray crystallography, NMR or CryoEM. To circumvent these obstacles and to obtain structural insight into the SMN-complex, the Schizosaccharomyces pombe SMN complex was used as a model system in this work. In a collaboration with the laboratory of Dr. Remy Bordonne (IGMM, CNRS, France), we could show that the SpSMN complex is minimalistic in its composition, consisting only of SpSMN, SpGemin2, SpGemin8, SpGemin7 and SpGemin6. Using biochemical experiments, an interaction map of the SpSMN complex was established which was found to be highly similar to the reported map of the human SMN complex. The results of this study clearly show that SpSMN is the oligomeric core of the complex and provides the binding sites for the rest of the subunits. Through biochemical and X-ray scattering experiments, the properties of the SpSMN subunit such as oligomerization viii and intrinsic disorder, were shown to determine the overall biophysical characteristics of the whole complex. The structural basis of SpSMN oligomerization is presented in atomic detail which establishes a dimeric SpSMN as the fundamental unit of higher order SpSMN oligomers. In addition to oligomerization, the YG-box domain of SpSMN serves as the binding site for SpGemin8. The unstructured region of SpSMN imparts an unusual large hydrodynamic size, intrinsic disorder, and flexibility to the whole complex. Interestingly, these biophysical properties are partially mitigated by the presence of SpGemin8•SpGemin7•SpGemin6 subunits. These results classify the SpSMN complex as a multidomain entity connected with flexible linkers and characterize the SpSMN subunit to be the central oligomeric structural organizer of the whole complex. / Die Biogenese von spliceosomalen UsnRNPs ist ein hochkomplexer zellulärer Prozess, der sowohl im Zellkern als auch im Zytoplasma stattfindet. Ein Hauptteil dieses Prozesses ist der Aufbau des Sm-Kernpartikels, der aus einem ringförmigen Heptamer aus sieben Sm-Proteinen (SmD1 · D2 · F · E · G · D3 · B) besteht, die um ein einzelsträngiges RNA-Motiv (das auch als Sm-Stelle bezeichnet wird) der spliceosomalen U snRNAs gewickelt ist. Dieser Prozess findet hauptsächlich im Zytoplasma durch die sequenzielle Wirkung von zwei Biogenesefaktoren statt, den PRMT5 und den SMN-Komplexen. Der PRMT5-Komplex besteht aus den drei Proteinen PRMT5, WD45 und pICln und ist für die symmetrische Dimethylierung bestimmter Argininreste in den C-terminalen Schwänzen einiger Sm-Proteine verantwortlich. Die Wirkung des PRMT5-Komplexes führt zur Bildung von inkompetenten Sm-Protein-Intermediaten, die durch das Assemblierungs-Chaperon pICln (SmD1 · D2 · F · E · G · pICln und pICln · D3 · B) sequestriert werden. Aufgrund der Wirkung von pICln interagieren die Sm-Proteine in diesen Komplexen nicht mit den U snRNAs, um den reifen Sm-Kern zu bilden. Diese kinetische Falle wird durch die Wirkung des SMN-Komplexes aufgelöst, der die pICln-Untereinheit entfernt und die Bindung der Sm-Core-Zwischenprodukte an die U snRNA erleichtert, wodurch der reife Sm-Core-Partikel gebildet wird. Der menschliche SMN-Komplex besteht aus 9 Untereinheiten, die als SMN, Gemin2-8 und Unrip bezeichnet werden. Bisher sind keine atomaren Strukturen des gesamten SMN-Komplexes verfügbar, aber Strukturen isolierter Domänen und Untereinheiten des Komplexes wurden in den letzten Jahren von mehreren Laboratorien beschrieben. Der Mangel an strukturellen Informationen über den gesamten SMN-Komplex liegt höchstwahrscheinlich in den biophysikalischen Eigenschaften des SMN-Komplexes, der einen oligomeren SMN-Kern und viele unstrukturierte und flexible Regionen besitzt. Dies waren die größten Hindernisse für die Strukturaufklärung mit traditionellen Methoden wie Röntgenkristallographie, NMR oder CryoEM. Um diese Hindernisse zu umgehen und strukturelle Einblicke in den SMN-Komplex zu erhalten, wurde in dieser Arbeit der SMN-Komplex von Schizosaccharomyces pombe als Modellsystem verwendet. In Zusammenarbeit mit dem Labor von Dr. Remy Bordonne (IGMM, CNRS, Frankreich) konnten wir zeigen, dass der SpSMN-Komplex in seiner Zusammensetzung minimalistisch ist und nur aus SpSMN, SpGemin2, SpGemin8, SpGemin7 und SpGemin6 besteht. Mit biochemischer Experimenten wurde eine x Interaktionskarte des SpSMN-Komplexes erstellt, die der bekannten Karte des menschlichen SMN-Komplexes sehr ähnlich war. Die Ergebnisse dieser Studie zeigen deutlich, dass SpSMN der oligomere Kern des Komplexes ist und die Bindungsstellen für den Rest der Untereinheiten bereitstellt. Durch biochemische und Röntgenstreuungsexperimente wurde gezeigt, dass die Eigenschaften der SpSMNUntereinheit wie Oligomerisierung und intrinsische Störung die gesamten biophysikalischen Eigenschaften des gesamten Komplexes bestimmen. Die strukturelle Basis der SpSMN-Oligomerisierung wird atomar detailliert dargestellt, wodurch ein dimeres SpSMN als zentrale Grundeinheit der SpSMN-Oligomere höherer Ordnung festgelegt wird. Zusätzlich zur Oligomerisierung dient die YG-Box- Domäne von SpSMN als Bindungsstelle für SpGemin8. Die unstrukturierte Region von SpSMN verleiht dem gesamten Komplex eine ungewöhnlich große hydrodynamische Größe, intrinsische Unordnung und Flexibilität. Interessanterweise werden diese biophysikalischen Eigenschaften teilweise durch das Vorhandensein von SpGemin8 • SpGemin7 • SpGemin6-Untereinheiten gemindert. Diese Ergebnisse klassifizieren den SpSMN-Komplex als eine mit flexiblen Wechselwirkungen verbundene Multidomäneneinheit und charakterisieren die SpSMN-Untereinheit als den zentralen oligomeren Strukturorganisator des gesamten Komplexes.

Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2009-2010 / Les cassures double-brin de l'acide désoxyribonucléique (ADN) sont parmi les lésions les plus cytotoxiques car une seule lésion non réparée est létale chez la levure. Dans le but de réparer efficacement ces dommages, la cellule dispose de différents mécanismes tels que le Non Homologous End Joining (NHEJ). Toutefois ces mécanismes peuvent causer des mutations et, lorsqu' il est possible, la cellule privilégie un système qui permet une réparation très fiable: la recombinaison homologue. Ce processus peut aussi être utilisé lors de la méiose pour créer de la diversité génétique. La méiose est un mécanisme complexe et une mauvaise régulation peut mener à des problèmes tels que l' aneuploïdie. La recombinaison homologue méiotique se divise en quatre étapes majeures: (i) l' initiation qui crée des cassures double-brin par un complexe muItiprotéique incluant Rec12; (ii) la résection de l'ADN ou les protéines majeures sont Rad32/Rad50/Nbsl; (iii) l'invasion d'un duplex homologue avec les protéines RadSl et Dmcl, et pour terminer (iv) la résolution des jonctions de Holliday. Lors de mes études de doctorat, je me suis concentré sur la caractérisation biochimique des principales protéines des trois premières étapes (initiation, résection et particulièrement l'invasion) de la recombinaison méiotique chez Schizosaccharomyces pombe permettant la réparation des cassures double-brin. Mes travaux avaient pour but de caractériser les protéines Dmcl et le complexe Hop2/Mndl chez S.pombe. Nous avons déterminé que Dmcl avait comme Rad 51 la capacité de former un filament hélical sur l'ADN simple brin, ansi que de catalyser des réactions d'échange de brin. D'autre part j'ai mis en évidence le rôle que joue le complexe Hop2/Mndl dans la stimulation de Dmc 1, ainsi que les différences entre les protéines de S.pombe et de la SOurIS.