Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains ix, 172 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Η χρήση της βιοπληροφορικής σε βιολογικά δεδομένα υψηλής απόδοσης είναι μια πολλά υποσχόμενη προσέγγιση για τη δημιουργία νέα γνώσης. Στην παρούσα εργασία αναλύεται ένα σύνολο δεδομένων που αφορά στα σημεία έναρξης της αντιγραφής (αφετηρίες) στο ζυμομύκητα Schizosaccharomyces pombe, όπως αναγνωρίστηκαν από πειράματα μικροσυστοιχιών που κάλυπταν όλο το μήκος του γονιδιώματος του οργανισμού (full genome). Οι αντιγραφή ξεκινάει από μεγάλο αριθμό αφετηριών οι οποίες βρίσκονται διάσπαρτες σε όλο το γονιδίωμα και μέχρι τώρα οι περισσότερες μελέτες των χαρακτηριστικών των αφετηρίων είχαν πραγματοποιηθεί για περιορισμένο αριθμό αυτών. Στην εργασία αυτή αναλύονται για πρώτη φορά τα χαρακτηριστικά του συνόλου των αφετηριών του S. pombe με σκοπό να διαπιστωθεί ποια χαρακτηριστικά καθορίζουν πότε μια περιοχή του γονιδιώματος μπορεί να δράσει ως αφετηρία αντιγραφής. Από την ανάλυση αυτή προκύπτει ότι: 1. Οι αφετηρίες έχουν υψηλότερο μέγιστο περιεχόμενο ΑΤ από άλλες γονιδιωματικές περιοχές. 2. Οι αφετηρίες εντοπίζονται κατά προτίμηση σε μεγάλες διαγονιδιακές περιοχές ανάμεσα σε αποκλίνουσες μεταγραφικές μονάδες. 3. Η ασυμμετρία κατανομής Α και Τ ενδέχεται να αποτελεί δείκτη των αφετηριών. 4. Η απόδοση έχει συσχέτιση με το περιεχόμενο ΑΤ. / Use of Bioinformatics in high-throughput biological data is a promising approach for creation of new knowledge. In this work we analyze a dataset that concerns origins of DNA replication initiation in the yeast Schizosaccharomyces pombe, that were identified through full genome microarray experiments. DNA replication starts from a large number of origins that span the entire genome and until recently most studies of origins of replication have been carried out only for a limited number of them. Here we analyze for the first time the properties of the entire dataset of origins of replication in S. pombe in order to find out which specific properties define which genomic location can function as an origin of replication. From this analysis we found that: 1. Origins of replication have higher maximum AT (adenine-thymine) content than other genomic locations. 2. Origins of replication are found preferentially in large genomic locations between divergent transcriptional units. 3. AT asymmetry might be a marker of origins of replication. 4. The origin of replication firing efficiency is correlated with AT content.

DNA damage or replication stress during S-phase can activate the S-phase checkpoint which executes a variety of responses, such as the inhibition of origin firing and replication fork stabilisation. Deregulation of the S-phase checkpoint leads to genomic instability, which has been implicated in diseases such as cancer. In this thesis, I aimed to address whether the S-phase checkpoint is regulated outside of S-phase, and how the S-phase checkpoint targets its substrates in budding yeast. Although this checkpoint has thus far been associated exclusively with S-phase, it remains unknown whether its responses such as inhibition of origin firing can also occur in other phases of the cell cycle. To investigate this, the targets of the S-phase checkpoint for the inhibition of origin firing were analysed outside of S-phase upon DNA damage. Interestingly, I showed that the S-phase checkpoint effector kinase Rad53 phosphorylates its targets to inhibit origin firing outside of S-phase upon DNA damage when there is no replication. I then set out to test whether inhibition of origin firing by Rad53 outside of S-phase might be important for faithful DNA replication. Having shown that the checkpoint response is not specific for any cell cycle phases, I then tested how the specificity of Rad53 for its substrates might be determined. After demonstrating that the essential replication protein Cdc45 is required for Rad53 to phosphorylate the initiation factor Sld3, the key residues of Cdc45 necessary for Rad53 interaction were identified. A Cdc45 allele was produced by mutating the identified residues. This allele of Cdc45 is a separation-of-function mutant which prevents Sld3 phosphorylation upon DNA damage, but retains its function in DNA replication. Because Cdc45 travels with the replication fork, it is possible that Cdc45 also targets Rad53 to the replication fork to stabilise it upon replication stress. Overall, this thesis provides evidence that the S-phase checkpoint can function throughout the cell cycle and that Cdc45 targets Rad53 to some of its substrates, and possibly plays a role in replication fork stabilisation.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第18333号 / 農博第2058号 / 新制||農||1023(附属図書館) / 学位論文||H26||N4840(農学部図書室) / 31191 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 奥野 哲郎, 教授 佐久間 正幸, 准教授 吉田 天士 / 学位規則第4条第1項該当

Distributed real-time databases can be used to support data sharing for applications in wireless ad-hoc networks. In such networks, topology changes frequently and partitions may be unpredictable and last for an unbounded period. In this thesis, the existing database replication protocol PRiDe is extended to handle such long-lasting partitions. The protocol uses optimistic and detached replication to provide predictable response times in unpredictable networks and forward conflict resolution to guarantee progress. The extension, pPRiDe, combines update and state transfer strategies. Update transfer for intra-partition communication can reduce bandwidth usage and ease conflict resolution. State transfer for inter partition conflicts removes dependency on a common state between partitions prior to the merge to apply update messages on. This makes the resource usage independent of the life span of partitions. This independence comes at the cost of global data stability guarantees and pPRiDe can thus only provide per partition guarantees. The protocol supports application specific conflict resolution routines for both state and update conflicts. A basic simulator for mobile ad-hoc networks has been developed to validate that pPRiDe provides eventual consistency. pPRiDe shows that a hybrid approach to change propagation strategy can be beneficial in networks where collaboration by data sharing within long lasting partitions and predictable resource usage is necessary. These types of systems already require the conflict management routines necessary for pPRiDe and can benefit from an existing protocol. In addition to pPRiDe and the simulator this thesis provides a flexible object database suitable for future works and an implementation of PRiDe on top of that database.

L’information génétique contenue dans le noyau de la cellule doit être dupliquée fidèlement afin d’être transmise aux cellules filles pendant la division cellulaire. Pour organiser leur division, les cellules suivent un cycle reproductible composé de quatre étapes appelé cycle cellulaire. La préparation et l’exécution du programme de réplication de l’ADN ont lieu pendant des phases spécifiques du cycle grâce à l’intervention de multiples partenaires protéiques et de régulateurs structuraux. En particulier, la réplication de l’ADN s’effectue sur une matrice complexe constituée d’ADN associé à des protéines appelée chromatine. Cette dernière influence et est influencée par la réplication de l’ADN. Le travail présenté ici a pour objectif de faire le lien entre la conformation de la chromatine et la cinétique de réplication de l’ADN. Pour ce faire, nous combinons plusieurs techniques. La cytométrie de flux nous permet de suivre la quantité d’ADN présent dans une population de cellules et, à l’aide d’une méthode développée dans notre laboratoire, d’extraire le programme de réplication moyen d’une population de cellules. La technique de SAXS fournit des informations sur l’organisation locale des protéines et de l’ADN in vivo. Nos données peuvent être interprétées comme un cristal liquide avec un ordre nématique et une faible longueur de corrélation, ce qui suggère que la chromatine de la levure est majoritairement dépourvue d’une organisation en fibre de 30nm in vivo. Par ailleurs, par la méthode de peignage d’ADN, nous reproduisons les résultats précédemment obtenus montrant que la distance entre zones répliquées est d’environ ~60kb qui correspond à la distance entre des origines de réplication identifiées. Cependant, d’après l’étude du comportement dynamique de l’initiation, nous proposons que les initiations sont plus fréquentes que ce qui a été mesuré précédemment et correspondent à la distance entre les protéines MCM disposées sur le génome. / Genetic information carried in the cell nucleus must be faithfully duplicated to be transmitted to daughter cells during cell division. In order to orchestrate their division, cells go through a reproducible 4 stages cycle called «cell cycle». The preparation and execution of the DNA replication program is restricted to specific phases and implies many proteic and structural regulators. In particular, DNA replication occurs on a complex template of DNA associated with proteins. The latter is both influencing and influenced by DNA replication. This work aims at investigating the link between chromatin conformation and the kinetics of DNA replication. In order to do so, we combine several techniques. Using flow cytometry, we follow the evolution of a cell population with regards to their DNA content and, with a method developed in our laboratory, decipher the population averaged temporal program of DNA replication. SAXS data provide information on the local organisation of protein and DNA in vivo. Our data can be interpreted as a liquid crystal with a nematic order and a short correlation length, which suggest that yeast chromatin in vivo is predominantly devoid of 30 nm fibres organisation. On the other hand, we performed DNA combing to study the replication program in single cells. We reproduce previously obtained result showing that distance between replicated tracks is of ~60kb which corresponds to the distance between known origins of replication. However, studying the behaviour of initiation, we propose that the initiation events are more frequent than previously measured and correspond to distances between MCMs proteins loaded on the genome.

In order to monitor the T7 replisome fate upon encountering abasic lesion, I optimized a single molecule flow stretching assay where the replisome encounters either abasic site or undamaged site inserted at 3.5 kilobases from the replication fork. The obtained events were categorized into three groups; bypass, restart and permanent stop. The results showed 52% bypass, 39% pause and 9% stop upon encountering the abasic lesion. The pause duration in the restart events was found to be ten times longer than the undamaged one. Moreover, an ensemble experiment was performed, and the results were slightly consistent with regard to the bypass percentage (70%) but the stoppage percentage was significantly higher in the ensemble replication reaction (30%). Further investigations were made and it was found that the rate of the T7 replisome increases after bypassing the abasic lesion. To inquire more about this rate switch and the difference between the single molecule and ensemble results, another unwinding experiment was performed where only gp4 (helicase) was used from the replisome. Interestingly, the rate of DNA unwinding by gp4 was similar to the rate observed after the replisome bypasses the lesion. We hypothesize that the polymerase is stalled at the abasic site and its interaction with the helicase is lost. Consequently, the helicase and the polymerase will uncouple where the helicase continues unwinding the DNA to result in a higher observed rate after bypassing the abasic site. Additional studies will be performed in the future to directly observe the helicase and polymerase uncoupling upon encountering the lesion.

Indiana University-Purdue University Indianapolis (IUPUI) / Accurate DNA replication is vital for maintaining genomic stability. Consequently, the machinery required to drive this process is designed to ensure the meticulous maintenance of information. However, random misincorporation of errors reduce the fidelity of the DNA and lead to pre-mature aging and age-related disorders such as cancer and neurodegenerative diseases. Some of the incorporated errors are the result of the error prone DNA polymerase alpha (Pol α), which initiates synthesis on both the leading and lagging strand. Lagging strand synthesis acquires an increased number of polymerase α tracks because of the number of Okazaki fragments synthesized per round of the cell cycle (~50 million in mammalian cells). The accumulation of these errors invariably reduces the fidelity of the genome. Previous work has shown that these pol α tracks can be removed by two redundant pathways referred to as the short and long flap pathway. The long flap pathway utilizes a complex network of proteins to remove more of the misincorporated nucleotides than the short flap pathway which mediates the removal of shorter flaps. Lysine acetylation has been reported to modulate the function of the nucleases implicated in flap processing. The cleavage activity of the long flap pathway nuclease, Dna2, is stimulated by lysine acetylation while conversely lysine acetylation of the short flap pathway nuclease, FEN1, inhibits its activity. The major protein players implicated during Okazaki fragment processing (OFP) are known, however, the choice of the processing pathway and its regulation by lysine acetylation of its main players is yet unknown. This dissertation identifies three main findings: 1) Saccharomyces cerevisiae helicase, petite integration frequency (Pif1) is lysine acetylated by Esa1 and deacetylated by Rpd3 regulating its viability and biochemical properties including helicase, binding and ATPase activity ii) the single stranded DNA binding protein, human replication protein A (RPA) is modified by p300 and this modification stimulates its primary binding function and iii) lysine acetylated human RPA directs OFP towards the long flap pathway even for a subset of short flaps.

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