Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 106-115).

Precancerous lesions from a variety of human tissues display markers of DNA damage suggesting that genetic instability occurs early during the process of carcinogenesis. Consistent with this, several oncogenes can activate ATM and other components of the DNA damage response pathway when expressed in cultured cells. Here we demonstrate that preneoplastic epithelial tissues from four different transgenic mouse models expressing the oncogenes c-myc, SV40 T antigen, human papilloma virus (HPV) E7, or E2F3a display [gamma]-H2AX foci and other markers of DNA damage. Moreover, transgenic expression of these oncogenes leads to increased levels of damaged DNA as measured by the comet assay. In at least the Myc transgenic model, the formation of [gamma]-H2AX foci is dependent on functional ATM. Inactivation of Atm also impairs p53 activation and reduces the level of apoptosis observed in transgenic tissue overexpressing Myc. This correlates with accelerated tumor development in Myc transgenic mice lacking ATM. To understand the mechanism by which oncogenes induce DNA damage, we employed an adenoviral overexpression system. Under conditions in which Myc or E2F3a induced replication is inhibited, we see a reduction in the DNA damage induced by these oncogenes both by comet assay and levels of [gamma]-H2AX. Moreover, Myc and E2F3a induced increased levels of the Cdt1 protein, a replication origin- licensing factor implicated in aberrant DNA replication. Taken together, these findings suggest that deregulated oncogenes induce unscheduled DNA replication leading to DNA damage and activation of the ATM DNA damage response pathway, which is important for the activation of p53, induction of apoptosis and the suppression of tumorigenesis. / text

abstract / Biochemistry / Master / Master of Philosophy

The "DNA Damage Hypothesis" pertaining to the evolution of sex was tested using Hydra littoralis. DNA damage was produced by irradiating whole live hydra with ultraviolet light. A curve of uv light dosage vs. survival was constructed. Estimations of threshold fluence and LD50 were made from the survival curve. In four separate experiments, using various combinations of environmental temperatures, uv doses, and number of doses, frequencies of asexual and sexual reproduction were observed and compared. The hydra that received uv treatments did not show an increase in the consequent amount of sexual reproduction, and actually showed a decrease. An increase in the amount of sexual reproduction following DNA damage is predicted by the DNA damage hypothesis, therefore these results do not support this theory. The data was also used to make contradictory observations regarding the "stress hypothesis" for the occurrence of sexual reproduction in hydra.

Colon cancer is the second most common type of cancer in the United States. Its incidence is linked epidemiologically to high levels of bile acids in the feces. Bile acids have been implicated as promoters and cocarcinogens in the etiology of colon cancer and as comutagens and mutagens in bacteria. These observations suggest the hypothesis that bile acids may damage DNA. By using the DNA-damage inducible SOS system in Escherichia coli, this study shows that when bacteria are exposed to bile acids there is induction of the SOS repair system and preferential survival of cells undergoing repair. Additionally, differential killing assays using repair defective bacteria show strains defective in recombinational repair or excision repair have lower survival when treated with bile acids than their parental wild-type counterparts. Human fibroblasts were treated with bile acids and unscheduled DNA synthesis (UDS) was measured. UDS is considered to represent the DNA synthesis step in excision repair. UDS, measured by autoradiography, was found to significantly increase in human fibroblasts upon treatment with bile acids. In addition, differential cytotoxicity assays with Chinese Hamster Ovary cells showed that different DNA-repair pathway defective cells were sensitive to different bile acids. Introduction of DNA damage and induction of DNA-repair by bile acids implicates them as possible direct carcinogens in the etiology of colon cancer.

Protein homo-oligomerization is the process through which identical peptides bind together to form higher order complexes. Self-interactions in many cases are constitutive and stable, used as building blocks for biological structures, such as rings, filaments and membranes. Further, homo-oligomerization can also be a regulatory process that influences the proteins' function such as change in transcriptional activities for transcription factors. Innovative methods to measure oligomerization in live cells are needed in order to understand regulation and function of homooligomerization in the native cellular context. This thesis examines the case of the tumor suppressor p53, whose homo-tetramerization greatly influences its activity as a transcription factor. We develop methods to quantify p53's self-interaction in individual living cells and follow it in time after DNA damage. The two methods we developed have complementary qualities and different applications. We first use fluorescent correlation spectroscopy to study the molecular events occurring in the first three hours of the p53 in response to double strand breaks. We find that in the absence of stress p53 is present in a mixture of, monomers, dimers and tetramers. When damage is sensed, oligomerization is rapidly induced and nearly all p53 is found bound in tetramers. We combine our data with a mathematical framework to propose the existence of a dedicated mechanism triggering p53 oligomerization independently of protein stabilization. Next, we use bimolecular fluorescent complementation to probe for tetramerization in the longer timescales of p53's response to ultraviolet radiation. In this context we find that even though the rate of p53 accumulation increases with the dose of radiation, p53 tetramers are formed at a steady rate. We hence propose the existence of an inhibitory mechanism that prevents the oligomerization reaction from following a linear input-output relation. We identify ARC, a known cofactor of p53, as part of this inhibitory mechanism. Downregulation of ARC restore the linear relation between to total and tetrameric p53. Finally, in both experimental setups higher oligomerization lead to an increase in p53 activity, underscoring the connection between regulation of oligomerization and the transcriptional activity of p53 in cancer cells. Collectively, this work emphasizes the importance of precise measurements to investigate the regulation and function of higher order complexes and provides generally applicable methods to quantify homo-oligomerization in live single cells.

Η αδειοδότηση της αντιγραφής αποτελεί μία ιδιαίτερα σημαντική διαδικασία, η οποία λαμβάνει χώρα στις θέσεις έναρξης της αντιγραφής με το σχηματισμό του προ-αντιγραφικού συμπλόκου, που περιλαμβάνει το ORC, το Cdc6, το Cdt1 καθώς και τις MCM 2-7. Η συγκεκριμένη διαδικασία, πραγματοποιείται μία φορά σε κάθε κυτταρικό κύκλο, κατά τη μετάβαση από την M φάση στη G1. Οι παράγοντες που συμμετέχουν υπόκεινται σε εκτεταμένη ρύθμιση προκειμένου να εξασφαλιστεί η απουσία επαναντιγραφής και γονιδιωματικής αστάθειας. Η πρωτεόλυση μορίων που συμμετέχουν στον κυτταρικό κύκλο αποτελεί έναν από τους μηχανισμούς που χρησιμοποιούν τα κύτταρα για τη ρύθμιση των επιπέδων διαφόρων παραγόντων. Το CRL4Cdt2 αποτελεί μία Ε3 λιγάση της ουβικουϊτίνης, η οποία είναι επιφορτισμένη με τη ρύθμιση των επιπέδων του αδειοδοτικού παράγοντα της αντιγραφής Cdt1 κατά την S φάση καθώς και μετά από βλάβες στο γενετικό υλικό. H συγκεκριμένη διαδικασία έχει διαπιστωθεί πως λαμβάνει χώρα σε όλα τα μετάζωα, καθώς και στον σχιζοσακχαρομύκητα. Μεταξύ των υποστρωμάτων του Cdt2 περιλαμβάνονται επίσης το Set8 και το p21. Στην παρούσα διπλωματική εργασία, πραγματοποιήθηκε μελέτη των περιοχών της πρωτεΐνης Cdt2 που είναι υπεύθυνες για τη στρατολόγηση της στην περιοχή της εντοπισμένης βλάβης στο γενετικό υλικό. Η μελέτη πραγματοποιήθηκε σε καρκινικά κύτταρα MCF7 που υπέστησαν εντοπισμένη βλάβη στο γενετικό υλικό με UV-C ακτινοβόληση και χρήση ειδικών πολυκαρβονικών φίλτρων με μικροπόρους. Για τη συγκεκριμένη μελέτη χρησιμοποιήθηκαν φορείς που εκφράζουν το αμινο-τελικό (2-417αα) και το καρβοξυ-τελικό (390-730αα) άκρο της πρωτεΐνης, καθώς και ένα μετάλλαγμα ολόκληρης της πρωτεΐνης όπου 6 SQ θέσεις που εντοπίζονται καρβοξυ-τελικά έχουν μεταλλαχθεί σε αλανίνες και δεν μπορούν να φωσφορυλιωθούν από τις ATM/ATR κινάσες. Μετά τη διεξαγωγή των πειραμάτων συσσώρευσης, διαπιστώθηκε πως στην περιοχή της εντοπισμένης βλάβης στρατολογούνται μόνο το Cdt2 6A(SQ) και Cdt2 (390-730). Ωστόσο, η συσσώρευση τους παρουσιάζεται ελαφρώς ασθενέστερη σε σχέση με την πρωτεΐνη αγρίου τύπου. Η συγκεκριμένη παρατήρηση καταδεικνύει πως στην περίπτωση του Cdt2 6A(SQ) η φωσφορυλίωση στις συγκεκριμένες SQ θέσεις είναι σημαντική για τη συσσώρευση της πρωτεΐνης στην περιοχή της βλάβης. Η στρατολόγηση του Cdt2 (390-730) στην περιοχή της βλάβης, συνιστά μία σημαντική παρατήρηση, η οποία υποδεικνύει ότι η πρωτεΐνη Cdt2 διατηρεί την ικανότητα συσσώρευσης στην περιοχή της βλάβης, παρά την απουσία των αλληλουχιών που βρίσκονται στο αμινο-τελικό άκρο και συμμετέχουν στην αλληλεπίδραση με τα υποστρώματα αλλά και στο σχηματισμό του συμπλόκου της λιγάσης. Προκειμένου να μελετηθεί η κινητική στην περιοχή της εντοπισμένης βλάβης των μεταλλαγμάτων που παρουσιάζουν συσσώρευση, χρησιμοποιήθηκε η τεχνική της επαναφοράς φθορισμού μετά από φωτολεύκανση (FRAP). Τα αποτελέσματα, κατέδειξαν πως τόσο στα κύτταρα χωρίς βλάβη, αλλά και σε αυτά με εντοπισμένη βλάβη υπάρχει παρόμοια κινητική μεταξύ της πρωτεΐνης αγρίου τύπου και των δύο μεταλλαγμάτων. Έτσι, η ύπαρξη μεταλλάξεων σε 6 SQ θέσεις στην περίπτωση του Cdt2 6A(SQ), αλλά και η απώλεια των αμινοτελικών αλληλουχιών στο Cdt2 (390-730) δεν μεταβάλλουν την κινητική στην περιοχή της βλάβης. Τέλος, έγινε μελέτη της συμβολής της φωσφορυλίωσης από τις ATM/ATR κινάσες, στη συσσώρευση στην περιοχή της βλάβης. Γι’ αυτό το λόγο έγινε χρήση της καφεΐνης, που αποτελεί αναστολέα των συγκεκριμένων κινασών. Τα αποτελέσματα, κατέδειξαν πως υπάρχει πολύ ασθενέστερη συσσώρευση στη συγκεκριμένη περίπτωση σε σχέση με την πρωτεΐνη αγρίου τύπου και το Cdt2 6A(SQ). Με αυτό τον τρόπο υποδεικνύεται πως η φωσφορυλίωση σε όλες τις SQ θέσεις του μορίου, είναι σημαντική για τη συσσώρευση του στην περιοχή της βλάβης. / Replication licensing is a crucial process which takes place at the origins of replication. It involves the formation of the pre-replicative complex consisting of ORC, Cdc6, Cdt1 and MCM 2-7. This process takes place during the late M to early G1 transition and happens only once per cell cycle. The tight regulation of the factors that participate in replication licensing prevents rereplication and genomic instability. Proteolysis is a central mechanism of the cell cycle which is important for the regulation of several factors. CRL4Cdt2 is an E3 ubiquitin ligase important for the regulation of the licensing factor Cdt1 during S phase and post DNA damage. It is known that the ubiquitination of Cdt1 by Cdt2 happens in all metazoans and in fission yeast. Set8 and p21 are also substrates of Cdt2. In this study, we investigated the domains of Cdt2 which are important for its recruitment at sites of localized DNA damage. To this end, we employed MCF7 cancer cells which were UV irradiated with polycarbonate filters with micropores. We used plasmids containing N-terminal (2-417aa) and C-terminal (390-730aa) constructs of the protein and a mutant of the entire protein containing alanine substitutions in 6 SQ sites located at the C-terminus of the protein which cannot be phosphorylated by the ATM/ATR kinases. The recruitment experiments indicated that only in the case of Cdt2 (390-730) and Cdt2 6A(SQ) there is recruitment at the site of localized DNA damage. However the recruitment in both cases was weaker compared to the wild type protein. In the case of Cdt2 6A(SQ) this observation indicates the importance of the phosphorylation of the six SQ sites for the recruitment of the protein at the site of damage. Cdt2 (390-730) recruitment indicates that Cdt2 can still get recruited at the site of damage even though it lacks motifs important for the recognition of the substrate and the formation of the ligase complex. In order to study the kinetics of the protein constructs that exhibit recruitment at the site of localized DNA damage we employed Fluorescence Recovery After Photobleaching (FRAP). The results showed that the kinetics of the constructs were similar to the wild type in undamaged cells and in cells with localized damage. This shows that the lack of phosphorylation of 6 SQ sites of Cdt2 6A(SQ) and the loss of the N-terminal motifs of Cdt2 (390-730) are not sufficient to cause any difference in the kinetics at the site of damage. Finally, we wanted to investigate the importance of the phosphorylation by the ATM/ATR kinases for the recruitment of Cdt2 at the sites of localized DNA damage. To this end, we employed the ATM/ATR inhibitor caffeine and we tracked the recruitment at the site of damage. The results showed that the recruitment was weaker compared to the wild type Cdt2 and the Cdt2 6A(SQ). This indicates the importance of the phosphorylation of the 9 SQ sites across the protein for the recruitment at the site of damage.

DNA damage tolerance (DDT) is a process utilized by cells to bypass replication blocking lesions in the DNA, preventing replication fork collapse and maintaining genomic stability and cell viability. In Saccharomyces cerevisiae DDT consists of two branched pathways. One branch allows direct replication past lesions in the DNA utilizing specific error-prone polymerases, a process known as translesion DNA synthesis (TLS). The other branch utilizes homologous recombination and template switch to replicate past damaged DNA in an error-free manner. RAD5 has traditionally been characterized as belonging to the error-free pathway of DNA damage tolerance. The protein is multi-functional, with several specific activities identified and classified to the error-free branch of DDT. However, there is also evidence for additional uncharacterized activities of the protein. The goal of this research was to determine which branches of DNA damage tolerance the uncharacterized activities of Rad5 are involved in. A two-pronged approach was utilized, elucidation of the physical interactions of the protein, and examination of the genetic interactions between RAD5 and other DDT genes. The evidence indicates that Rad5 plays a partial role in TLS and the protein is known to physically interact with Rev1, a member of the TLS pathway. We assumed this physical interaction mediates the TLS activity of Rad5. The yeast two-hybrid assay was utilized to examine the interaction between Rev1 and truncated Rad5 fragments, and the N-terminal 30 amino acids of Rad5 proved sufficient to maintain the interaction. This research sets the stage to identify key residues in Rad5 for the interaction with Rev1, and the creation of a TLS deficient rad5 mutant by targeting those key residues. Genetic interactions between RAD5 and genes required for the initiation of DDT in the cell were examined based on sensitivity to killing by various DNA damaging agents. We determined that the functions of Rad5 rely on PCNA modification, and thus do not function in a cellular process unrelated to Rad5. Potential uncharacterized functions are discussed on the basis of these results and the results of the interaction studies. Future structural and functional studies are proposed to better understand the role of Rad5 in the cell.

Oxidative stress and histone deacetylase (HDAC) inhibition has been implicated as potential mechanisms in valproic acid (VPA) teratogenicity. Reactive oxygen species (ROS) can target DNA to cause oxidative DNA damage and DNA double strand breaks (DSBs) which can be repaired through homologous recombination (HR). HR is not an error free process and can result in detrimental genetic changes. In this present study we evaluated the role of HDAC inhibition in VPA-initiated HR. HDAC inhibition may indirectly alter repair activity as a result of increased expression of genes involved in HR or indirectly by causing DNA damage which initiates repair. The first objective was to investigate the ability of VPA to cause HDAC inhibition in the Chinese hamster ovary (CHO) 33 cell line. Using immunblotting, an increase in acetylated histone H3 and H4 protein levels was observed throughout 24 hr exposure to 5 mM VPA. Secondly, to investigate whether VPA affects the activity of DNA DSB repair, CHO 33 cells were transfected with either the endonuclease I-SceI plasmid to induce a site specific DSB or the empty plasmid, pGem. However, no increase in the difference in HR between VPA and media exposed I-Sce1 transfected cells compared to cells transfected with pGem was observed, which suggests that VPA does not affect DNA repair activity. Thirdly, to determine if VPA-induced HDAC inhibition increases susceptibility to DNA damage, immunocytochemistry revealed an increase in the number of γ-H2AX foci throughout 24 hr exposure to 5 mM VPA. To determine if oxidative stress may play a role in mediating VPA-induced DNA DSBs, another recombination study was carried out in which cells were pretreated with 400 U/ml of PEG-catalase prior to VPA treatment. The observed protective effect of PEG-catalase against VPA-induced HR and the generation of intracellular ROS by VPA suggest ROS may also play a role in VPA-initiated HR. However, in our DNA oxidation study, no increase in the oxidized nucleosides, 8-hydroxy-2'-deoxyguanosine and 5-hydroxycytosine was observed after VPA treatment. These studies suggest that HDAC inhibition and ROS signalling may play other roles in DNA maintenance and cell cycle arrest in initiating DNA DSBs and HR repair. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2009-08-12 14:27:16.327