The in vivo synthesis of deoxyribonucleic acid from labeled precursors was studied in the rat intestinal mucosa in an attempt to elucidate the complex process of DNA replication. In one set of experiments, the rats were injected with ³H-thymidine and then starved for 24 hours, in which time the stable DNA became labelled with tritium. (14)C-thymidine was then administered and the animals were sacrificed 5 minutes later. By this procedure the newly synthesized DNA was labelled with (14)C. The DNA, was fractionated by chromatography on a methylated-albumin kieselguhr column. Only one main peak of DNA was eluted with a sodium chloride solution ranging in concentration from 0.5-0.6 M. The thermal denaturation temperature for the DNA in each.fraction from this peak was determined and the G + C content was calculated:, Within the DNA peak obtained from MAK chromatography, the G + C content of the DNA decreased with increasing fraction number. In addition to these differences in base composition, there were differences in metabolic activity between the fractions, which were indicated by their ³H/ (14)C ratios. The ³H/ (14)C ratio of the DNA fractions from MAK chromatography increased with fraction number to a maximum at fraction 4 or 5 and then decreased. It was found that the ³H/O.D. ratio of the fractions was not constant, thus suggesting that the tritium might be unevenly distributed throughout the fractions. If the time interval between the ³H and (14)C-thymidine injections was reduced to 3 ½ hours, the ³H/O.D. ratio became constant while the pattern of the ³H/14C ratios remained unchanged. If (14)C-thymidine was administered 20 minutes before the animals were sacrificed, the ³H/(14)C ratio of the DNA fractions from MAK chromatography increased with increasing fraction number. From these results it was concluded that small molecular weight, newly synthesized DNA, which was highly labelled with (14)C, was being incorporated with time into the high molecular weight, stable DNA fraction, which is labelled with ³H. During these experiments it was observed that the pattern of ³H/(14)C ratio versus fraction number varied according to the treatment given to the DNA sample prior to the preparation for radioactive counting. If the sample was denatured by heating to obtain its T(M) value, and then dialyzed against distilled water, small molecular weight nucleotides passed into the dialysate. The denatured DNA sample also gave different results from the native DNA sample on digestion with snake venom phosphodiesterase. On the denatured sample, the pattern of release of ³H and (14)C labelled material into the acid soluble material, indicated that both these labels were uniformly distributed along the DNA chain. On the other hand, with the native 5 min. DNA samples, the release of (14)C labelled material into the acid soluble fraction was that expected for DNA which had incorporated (14)C-preferentially into the 3’ terminal positions. The separation of the pyrimidine clusters of DNA indicated that those were not uniformly labelled with (14)C and ³H. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Replication Protein A (RPA), the eukaryotic single-stranded DNA-binding complex, is essential for multiple processes in cellular DNA metabolism including, but not limited to, DNA replication, DNA repair and recombination. The `canonical' RPA is composed of three subunits (RPA1, RPA2, and RPA3). In addition to the three canonical subunits, there is a human homolog to the RPA2 subunit, termed RPA4, which can substitute for RPA2 in complex formation. The resulting RPA complex has been termed `alternative' RPA (aRPA). The normal function of aRPA is not known; however, previous studies have shown that it does not support S-phase progression in vivo. The goal of this thesis was to characterize the function of aRPA in DNA replication, DNA repair and recombination and profile its expression in human tissues. The studies presented in this thesis show that the aRPA complex has solution and DNA binding properties indistinguishable from the canonical RPA complex as determined by gel mobility shift assays. However, aRPA was unable to support DNA replication and inhibited canonical RPA function in a cell-free simian virus 40 system. aRPA inhibited both initiation and elongation of DNA synthesis in the SV40 system. Two regions of RPA4, the putative L34 loop and the C-terminal winged helix domain, were responsible for inhibiting SV40 DNA replication. The mechanism of SV40 DNA replication inhibition during initiation and elongation was characterized using assays for DNA polymerase α and DNA polymerase δ. aRPA was shown to have reduced interaction with DNA polymerase α and was not able to efficiently stimulate DNA synthesis by DNA polymerase α on aRPA coated single-stranded DNA. However, aRPA stimulated DNA synthesis by DNA polymerase δ in the presence of PCNA and RFC even though a reduced interaction was observed between aRPA and polymerase δ. The role of aRPA in DNA repair was also investigated. aRPA interacted with both Rad52 and Rad51 but had a reduced interaction with Rad51. However, aRPA was still able to stimulate Rad51-dependent strand exchange. aRPA also supported the dual incision/excision reaction of nucleotide excision repair. aRPA was less efficient in nucleotide excision repair than canonical RPA and this reduction was attributed to reduced interactions with the repair factor XPA. In contrast, aRPA exhibited higher affinity for damaged DNA than canonical RPA. The expression of RPA4 and RPA2 was determined by quantitative PCR in established cell lines, human normal tissues and human tumor tissue. RPA4 was shown to be expressed in all normal tissues examined but the level of expression was tissue specific. Additionally, RPA4 expression was decreased in all tumor tissues examined and was at the limit of detection in established cell lines. Taken together, the results presented in this thesis suggest that aRPA is a `non-proliferative' form of RPA that functions to maintain the genomic stability of non-dividing cells.

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.

No description available.

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.

Tato práce se zabývá buněčným cyklem kvasinky Saccgaromyces cerevisiae. Oblastí našeho zájmu je přechod mezi G1 a S fází, kde je naším cílem identifikovat velikosti buňky v době počátku DNA replikace. Nejprve se věnujeme nedávno publikovanému matematickému modelu, který popisuje mechanismy vedoucí k S fázi. Práce poskytuje detailní popis tohoto modelu, stejně jako časový průběh některých důležitých proteinů či jejich sloučenin. Dále se zabýváme pravděpodobnostním modelem aktivace replikačních počátků DNA. Nově uvažujeme vliv šíření DNA replikace mezi sousedícími počátky a analyzujeme jeho důsledky. Poskytujeme také senzitivní analýzu kritické velikosti buňky vzhledem ke konstantám popisujícím dynamiku reakcí v modelu G1/S přechodu.

In this thesis, I address two fundamental questions related to our understanding of how DNA damage is processed and repaired during replication. Using Two-dimensional (2-D) agarose gel analysis, I first examine whether DNA damage on plasmids introduced by transformation is processed in a manner similar to that observed on endogenously replicating plasmids and the chromosome. The original intent for using this approach was to develop a technique that could examine how different DNA adducts would be repaired in various sequence contexts. However, I found that distinct differences exist between the processing of DNA damage on transforming plasmids and the chromosome. The 2-D agarose gel analysis shows that RecA-mediated processing does not contribute to the survival of transforming plasmids and that this effect is likely due to inefficient replication of the plasmids after they are initially introduced into cells. These observations, while important, place limitations on the usefulness of transforming plasmids to characterize cellular repair processes. In a second question, I characterize the composition of the replisome following arrest by UV-induced DNA damage. Using 2-D agarose gel analysis the structural changes that occur in DNA during processing and repair have been well characterized, however, little is known about the fate of the replisome itself during these events. I used thermosensitive replication mutants to compare the DNA structural intermediates induced after disruption of specific components of the replisome to those observed after UV damage. The results show that dissociation of subunits required for polymerase stabilization are sufficient to induce the same processing events observed after UV damage. By contrast, disruption of the helicase-primase complex induces abnormal structures and a loss of replication integrity, suggesting that these components remain intact and bound to the template following replication arrest. I propose that polymerase dissociation provides a mechanism that allows repair proteins to gain access to the lesion while retention of the helicase serves to maintain the integrity and licensing of the fork so that replication can resume from the appropriate site once the lesion has been processed.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / Ends of linear chromosomes are maintained by specialized structures known as telomeres. These structures are protected by a number of essential protein complexes including the shelterin complex and CST (CTC1 – STN1 – TEN1) complex. CST is an RPA-like ssDNA binding protein that is vital for telomere length maintenance via inhibition of telomerase and stimulation of DNA polymerase α -primase during C-strand fill-in synthesis. CST is also known to possess additional genome-wide roles in regulating DNA replication and repair including helping facilitate replication re-start at stalled forks, activating checkpoint signaling at double-strand breaks, and promoting replication origin firing. Proper and efficient repair of DNA is critical in order to protect the integrity of the genome and prevent extreme mutagenesis. Telomeres have a strong predisposition to oxidative DNA damage in the form of 8-oxoguanine caused by exposure to reactive oxygen species and free radicals. These oxidative lesions are repaired by the base-excision repair (BER) pathway. Previous work has implicated telomeric proteins such as the shelterin complex in mediating BER. Here we show for the first time that the CST complex and individual subunits robustly stimulate a myriad of proteins involved in the BER pathway including Pol β, APE1, FEN1, and LIGI. CST’s ability to augment these BER-associated proteins could be instrumental in promoting efficient DNA repair. Additionally, we find that CTC1 and STN1 are able to significantly enhance the polymerase activity of Pol δ and Pol α on both random-sequence and telomeric-sequence DNA substrates in vitro. What is more, we establish the ability of CST to resolve G4 structure and promote Pol δ synthesis, which we predict is a key feature of CST’s involvement in DNA replication at telomeres, which are known to form replication-inhibiting G4’s. Our results define important mechanistic insight into CST’s role in DNA replication and repair, and provide a strong foundation for future studies relating defective telomere maintenance to aging disorders and cancers which impact human health.

No description available.