A trinucleotide repeat (TNR) is a 3 base pair (bp) DNA sequence tandemly repeated in an array. In humans, TNR sequences have been found to be associated with at least 14 severe neurological diseases including Huntington disease, myotonic dystrophy and several of the spinocerebellar ataxias. Such diseases are caused by an expansion of the repeat sequence beyond a threshold length and are characterized by non-Mendelian patterns of inheritance which lead to genetic anticipation. Although the mechanism of the genetic instability in these arrays is not yet fully understood, various models have been suggested based on the in vitro observation that TNR sequences can form secondary structures such as pseudo-hairpins. In order to investigate the mechanisms responsible for instability of TNR sequences, a study was carried out on Escherichia coli cells in which TNR arrays had been integrated into the chromosomal lacZ gene. This genetic assay was used to identify proteins and pathways involved in deletion and/or expansion instability. Deletion instability was clearly dependent on orientation of the TNR sequence relative to the origin of replication. Interestingly, it was found that expansion instability is not dependent on the orientation of the repeat array relative to the origin of replication. The replication fork reversal pathway and the RecFOR mediated gap repair pathway were found to have no statistically significant influence on the instability of TNR arrays. However, the protein UvrD was found to affect the deletion instability of TNR sequences. The roles of key helicase genes were investigated for their effects on instability of chromosomal CTG•CAG repeats. Mutation of the rep gene increased deletion in the CTG leading-strand orientation of the repeat array, and expansion in both orientations - destabilizing the TNR array. RecQ helicase was found to have a significant effect on TNR instability in the orientation in which CAG repeats were present on the leading-strand relative to the origin of replication. Mutation of the recQ gene severely limited the number of expansion events in this orientation, whilst having no effect on deletions. This dependence of expansions on RecQ was lost in a rep mutant strain. In a rep mutant expansions were shown to be partially dependent on the DinG helicase. All together, these results suggest a model of TNR instability in which expansions are due to events occurring at either the leading or lagging strand of an arrested replication fork, facilitated by helicase action. The identity of the helicase implicated is determined by the nature of the arrest.

Cancer cells take more glucose to provide energy and phosphoryl intermediates for cancer progression. Meanwhile, energy-provider function of mitochondria in cancer cells is disrupted. This phenomenon is so-called Warburg effect, which is discovered over eighty years ago. The detail mechanisms for Warburg effect are not well defined. How glycolytic enzymes contribute to cancer progression is not well known. PKM2 is a glycolytic enzyme dominantly localized in the cytosol, catalyzing the production of ATP from PEP. In this study, we discovered that there were more nuclear PKM2 expressed in highly proliferative cancer cells. The nuclear PKM2 levels are correlated with cell proliferation rates. According to our microarry analyses, MEK5 gene was upregulated in PKM2 overexpression cells. Our studies showed that PKM2 regulated MEK5 gene transcription to promote cell proliferation. Moreover, nuclear PKM2 phosphorylated Stat3 at Y705 site using PEP as a phosphoryl group donor to regulate MEK5 gene transcription. Our study also showed that double phosphorylated p68 RNA helicase at Y593/595 interacted with PKM2 at its FBP binding site. Under the stimulation of growth factors, p68 interacted with PKM2 to promote the conversion from tetrameraic to dimeric form so as to regulate its protein kinase activity. Overexpression PKM2 in less aggressive cancer cells induced the formation of multinuclei by regulating Cdc14A gene transcription. Overall, this study presents a step forward in understanding the Warburg effect.

P68 RNA helicase (p68), as a DEAD family protein, is a typical RNA helicase protein. P68 functions in many other biological processes, which include the regulations of the gene transcription, cell proliferation and cell differentiation. In our group, Y593 phosphorylated p68 was found to have a function in the epithelial mesynchymal transition, which is an important process for cancer metastasis. In the present study, we found that p68 is a nucleocytoplasmic shuttling protein. The protein carries two functional nuclear exporting signal sequences and two nuclear localization signal sequences. Calmodulin, a calcium sensor protein, is well known to play roles in cell migration by regulating the activities of its target proteins at the leading edge. Calmodulin interacts with p68 at the IQ motif of p68. However, the biological function of this interaction is not known. In this study, we found that the p68/calmodulin protein complex functions as a microtubule motor in migrating cells. The shuttling function of p68 along with the motor function of p68/calmodulin causes the leading edge distribution of calmodulin in migrating cells. Disruption the interaction between p68 and calmodulin inhibits cancer cell metastasis in an established mouse model. On the other hand, Y593-Y595 double phosphorylated p68 were found to interact with PKM2, an important tumor isoform of pyruvate kinase. The shuttling function of p68 is reasoned to promote the dimer formation of PKM2 and transport the PKM2 to the cell nucleus. The nuclear PKM2 was found to function as a protein kinase to promote cell proliferation. In specific, the nuclear PKM2 phosphorylates and activates Stat3, an important transcription factor functions in cell proliferation. Overall, p68 is found to have functions in both cell migration and cell proliferation, and these two functions depend on the nucleocytoplasmic shuttling activity and the post-translational modification of p68.

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No description available.

No description available.

The essential cell cycle kinase Dbf4/Cdc7 (DDK) triggers DNA replication through phosphorylation of the Mcm2-7 helicase at replication origins. Prior work has implicated various Mcm2-7 subunits as targets of DDK, however it is not well understood which specific subunits mediate the docking of the DDK complex. Through yeast two-hybrid and co-immunoprecipitation analyses, we found that Dbf4 and Cdc7 interact with distinct subunits of the Mcm2-7 helicase complex. Dbf4 showed the strongest interaction with Mcm2 while Cdc7 associated with Mcm4 and Mcm5. Dissection of the N-terminal region of Mcm2 revealed two regions that mediate the interaction with Dbf4, whereas in Mcm4, a region near the N-terminus has been previously identified by another group as the DDK docking domain. Mutant forms of Mcm2 (Mcm2ΔDDD) or Mcm4 (Mcm4ΔDDD) lacking the DDK docking domain were expressed in cells and resulted in modest growth and replication defects. Combining the two mutations resulted in synthetic lethality, suggesting a redundant mechanism of Mcm2 and Mcm4 in targeting the DDK complex to Mcm rings. Furthermore, growth inhibition could be induced in a Mcm4ΔDDD background by overexpressing Mcm2 to titrate Dbf4 from Mcm rings. These growth defects were exacerbated in the presence of genotoxic agents such as hydroxyurea and methyl methanesulfonate, suggesting that DDK-Mcm interactions may play a role in stabilizing replication forks under S-phase checkpoint conditions. Regions of Cdc7 were examined for their interaction with Mcm4 and Dbf4. Results have shown that the N-terminal amino acid region 55-124 and the C-terminal region 453-507 of Cdc7 are likely target regions for Dbf4-binding. Several conserved residues were identified within the N-terminal 55-124 Cdc7 region that interface with conserved residues within motif-C of Dbf4. Conserved residues were identified within the DDD domain of Mcm2 and mutating these residues resulted in a decreased interaction with Dbf4. Lastly, bioinformatics analysis has revealed potential conserved residues within the Mcm4DDD region, which may play a role in binding to Cdc7. This research is significant because these factors, which are conserved in all eukaryotes studied to date, should give further insight as to how DNA replication is triggered and how it is affected when cells are exposed to DNA damaging or replication compromising agents. This research also has implications in cancer genetics, as prior studies have shown elevated DDK and Mcm protein levels in tumour cell lines and melanomas, with Cdc7 showing great promise as a cancer therapeutic target. Such knowledge will further enhance our understanding of the DNA replication process and the roles of cell cycle proteins involved, under both normal and checkpoint conditions.

Es presenta el model atòmic a 4.5 Å de DnaB, la principal helicasa replicativa bacteriana, d'Aquifex aeolicus. És un anell hexamèric de 100 Å d'amplada i 80 Å d'alçada amb dues capes de simetria diferenciada, la dels dominis N-terminals en C3 i la dels C-terminals propera a C6. El diàmetre central és de 25 Å al llarg d'ambdues capes, principal diferència amb les estructures prèvies, on era 25 Å més estret a la capa N-terminal. L'estretament s'origina pel trencament d'una de les dues superfícies d'interacció entre monòmers N-terminals, cosa que augmenta la flexibilitat del subdomini implicat. Només l'ssDNA pot atravessar l'anell, quan a les estructures prèvies hi podia passar tant ssDNA com dsDNA. L'estructura aquí presentada és més propera a la conformació funcional de DnaB durant la realització de l'activitat helicasa, mentre que les anteriors correspondrien a la forma inactiva o a la conformació capaç de translocar-se sobre dsDNA. / DnaB is the main replicative helicase in bacteria. An atomic model for the DnaB from Aquifex aeolicus at a 4.5 Å resolution is presented. It´s a ring-shaped homohexamer (100 Å width and 80 Å hight) with two simmetry layers, a C3 N-terminal layer and an almost C6 C-terminal one. The diameter of the central channel is 25 Å along both layers, being the main diference with the previously solved structures, which were 25 Å smaller along the N-terminal layer. This is due to one of the previous interacting surphaces being lost in the current structure, thus enabling a higher felxibility of the subdomain involved. Only ssDNA can pass trhough the ring, while both ssDNA and dsDNA could in the previous structures. So, the present structure is closer to the functional conformation, while the previous ones would correspond to the inactive form or the conformation that is only able to translocate along dsDNA.

Heidelberg, Univ., Diss., 2008.

Les protéines Piwi sont exprimées dans les gonades (testicules et ovaires) des animaux où elles s'associent à des petits ARN nommés piRNAs (Piwi-interaction RNAs) et répriment les éléments transposables, défendant ainsi de l'intégrité du génome. En effet, les animaux knock-out pour les protéines Piwi montrent une perte de piRNAs et une activation des éléments transposables avec des conséquences catastrophiques: l'arrêt du développement des cellules germinales, due potentiellement à des dommages du génome qui entraîne l'infertilité. Le « Silencing » est réalisé soit par l'activité endonucléase guidée par les piRNAs de la protéine Piwi cytosolique ou par le recrutement de la machinerie de répression transcriptionelle sur les loci génomiques des cibles par Piwi nucléaire. La biogenèse des piRNAs peut être divisée en deux voies, une primaire et une secondaire. De longs ARN précurseurs simple-brin sont transformés en piRNAs matures de 25-30 nt qui sont chargés dans les protéines Piwi. Une fois chargée avec le piRNA, Piwi se lie aux transcrits de transposons ayant la séquence complémentaire et les clive en générant deux ARN, dont l'un peut être chargé dans une nouvelle protéine Piwi, produisant un piRNA secondaire. Des études génétiques ont identifié plusieurs facteurs protéiques essentiels à ce processus. Certain de ces facteurs sont des hélicases à ARN dont le rôle spécifique reste inconnu, principalement parce que ce sont des enzymes dynamiques et l'identification de leurs cibles et leurs partenaires protéiques avec des approches biochimiques standards est difficile.Dans la première partie de cette thèse nous décrivons comment l'introduction d'une mutation ponctuelle dans la boîte DEAD de l'hélicase à ARN Vasa (DEAD à DQAD) peut bloquer son activité in vivo et figer le complexe transitoire de biogenèse qui contient VASA et les deux protéines Piwi responsables de la biogenèse secondaire.La résolution de la structure de VASADQ en complexe avec l'ATP ou l'AMPPNP a révélé les détails moléculaires de cette inhibition et a expliqué le phénotype observé in vivo. VASADQ a un taux d'hydrolyse de l'ATP réduit, car après hydrolyse, le phosphate libre est bloqué à l'intérieur du site actif en raison d'une liaison hydrogène supplémentaire formée avec la Gln mutée. La réduction de l'hydrolyse de l'ATP se traduit par une faible liaison à l'ARN mesurée par des expériences biophysiques. L'introduction de la même mutation chez l'homologue murin de VASA (MVH) produit un phénotype de dominant négatif où MVHDQ est agglutinée sur le complexe RNP contenant les protéines Piwi et les piRNAs.Dans la deuxième partie de cette thèse, nous avons introduit la même mutation dans TDRD9, une autre hélicase à ARN impliquée dans la voie des piRNAs mais dont la fonction est inconnue. Nous avons d'abord exprimé, purifié TDRD9 et montré que la mutation dans son domaine hélicase DEVH à DQVH abolit complètement son activité ATPase sans impacter sur sa stabilité. Par la suite, nous avons généré une souris Knock-in et analysé son phénotype. Les souris Knock-in mâles sont stériles et présentent un blocage au début de la spermatogenèse qui est probablement une conséquence des dommages de l'ADN générés par l'activation des éléments transposables. Ces éléments, comme Line-1, présentent un défaut de méthylation à leur loci génomiques, mais qui ne semble pas être contrôlé par la voie piRNA dans le mutant, étant donné que les protéines Piwis sont correctement chargées avec les piRNAs dérivés de Line-1.Dans l'ensemble, nous avons étudié le rôle moléculaire de deux hélicases à ARN dans la voie des piRNAs, nous avons élucidé le rôle de VASA et nous montrons que l'activité ATPase de TDRD9 est essentielle pour la régulation des transposons au cours de la spermatogenèse de la souris. / PIWI proteins are expressed in the gonads (testis and ovary) of animals where they associate with PIWI-interacting RNAs (piRNAs) and silence transposable elements, defending the integrity of the genome. Indeed, animal knock-outs of Piwi proteins display a loss of piRNAs and activation of transposon sequences with catastrophic consequences: block in germ cell development potentially due to genome damage, resulting in infertility. Silencing is achieved either by piRNA-guided endonuclease activity of cytosolic Piwi protein or by recruitment of transcriptional repression machinery on target genomic loci by nuclear Piwi. Biogenesis of piRNAs can be divided in primary and secondary pathway. Primary pathway describes how long single-stranded RNA precursors are processed into mature 25-30 nt piRNAs and loaded into Piwi proteins. Piwi-loaded piRNAs bind and cleave complementary transposon transcripts generating two RNA products, one of which can be loaded into a new Piwi protein, generating a secondary piRNA. Different protein factors are essential in this process as identified by genetic studies. Few of these factors are putative RNA helicases but their specific role is unknown, mainly because RNA helicase are dynamic enzymes and identification of their targets and protein partners with standard biochemical approaches is challenging.In the first part of this thesis I describe how the introduction of a point mutation in the DEAD box of the RNA helicase Vasa (DEAD to DQAD) can block its activity in vivo and freeze a transient biogenesis complex that contains Vasa and the two Piwi proteins responsible for secondary biogenesis.Crystal structure of VASADQ in complex with ATP or AMPPNP revealed the molecular details of this inhibition and explained the phenotype observed in vivo. VasaDQ has a reduced ATP hydrolysis rate because after hydrolysis the free phosphate is blocked inside the active site due to an additional hydrogen bond formed with the mutated Gln. The reduction in ATP hydrolysis is mirrored by an impaired RNA binding activity as measured with biophysical experiments. Introduction of the same mutation in the mouse homologue of Vasa (MVH) has a dominant-negative phenotype where MVHDQ is clump on an ribonucleoprotein (RNP) complex containing piRNAs and mouse Piwi proteins.In the second part of this thesis I introduce the same mutation in TDRD9, another RNA helicase involved in piRNA pathway with an unknown function. First I expressed and purified TDRD9 and showed that DEVH to DQVH mutation in its helicase domain completely abolishes its ATPase activity but do not affects its stability. Next I created a knock-in mutation in the mouse genomic locus for Tdrd9 and analysed the resulting phenotype in the mutant. Knock-in mice are male sterile with an early block in spermatogenesis that is probably a consequence of uncontrolled DNA damages generated by de-repressed transposon elements. These elements, like Line-1, fail to be correctly methylated at their genomic loci in the Tdrd9 mutant. Although Tdrd9 is important for Line-1 transposon silencing, it is likely not via a role in piRNA biogenesis since Piwi proteins are correctly loaded with Line-1 derived piRNAs. Interestingly a drop in piRNAs that derives from SINE elements is observed in the mutant, probably reflecting a role for Tdrd9 in sorting primary transcripts into MILI and MIWI2 during DNA de novo methylation.Overall I investigated the molecular role of two RNA helicases in the piRNA pathway, elucidating the role of Vasa and show that the ATPase activity of Tdrd9 is essential for transposon regulation in mouse spermatogenesis.