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CHARACTERIZATION OF MUTL-MEDIATED PROTEIN INTERACTIONS IN DNA MISMATCH REPAIRPillon, Monica 07 October 2014 (has links)
DNA encodes the genetic information of the cell, therefore, every single living organism has a precise DNA damage response mechanism to safeguard DNA integrity. Base mismatches are endogenous DNA lesions introduced by the replicative polymerase during DNA replication. The conserved DNA mismatch repair pathway corrects these base mismatches. Mismatch repair initiation is orchestrated by two proteins, MutS and MutL. MutS recognizes and binds to base mismatches and relays the presence of the lesion to MutL. MutL, in turn, interacts with downstream factors to coordinate mismatch excision. The processivity clamp, typically known for its role in tethering the DNA polymerase to DNA during replication, is also involved in several steps of this repair process including MutL endonuclease activation and strand resynthesis. The dynamics of the MutS-MutL and MutL-processivity clamp interactions present one of the bottlenecks to uncovering the spatial and time organization of these protein assemblies. Therefore, little is known about the interactions that orchestrate the early steps of mismatch repair. The biochemical and structural work included in this thesis outlines a precise series of molecular cues that activate MutL. / Thesis / Doctor of Philosophy (PhD)
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Studying the Solution Behavior of DNA and DNA Sliding Clamps Using Various Fluorescence TechniquesJanuary 2013 (has links)
abstract: Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps. / Dissertation/Thesis / Ph.D. Chemistry 2013
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Mechanism of DNA Homologous Recombination through Studies of DNA Sliding Clamps, Clamp Loaders, and DNA PolymerasesLi, Jian 25 September 2013 (has links)
No description available.
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The molecular biology of DNA replication in the archaeon Sulfolobus solfataricusBeattie, Thomas R. January 2012 (has links)
DNA replication is essential for the propagation of all living organisms. The ability of a cell to accurately duplicate its entire genome is dependent upon the activity of numerous proteins. Identifying the molecular mechanisms by which these proteins act, and determining how they are physically and functionally coordinated at sites of active DNA replication, is central to understanding this essential cellular process. Archaea possess a DNA replication machinery which is ancestral to the one present in eukaryotes, and thus these organisms serve as simplified model systems for understanding the complexities of eukaryotic DNA replication. This thesis investigates the molecular mechanisms underlying Okazaki fragment maturation in the crenarchaeon Sulfolobus solfataricus, which is essential to the completion of lagging strand DNA replication. Reconstitution of Okazaki fragment maturation in vitro demonstrated that the activities of three enzymes – PolB1, Fen1, and Lig1 – are required for this process in S. solfataricus. Furthermore, it was shown that optimum coordination of their three distinct activities is dependent on the ability of PolB1, Fen1 and Lig1 to simultaneously interact with a single PCNA ring, providing evidence for a mechanism of multi-enzyme coordination which may be universally employed by DNA sliding clamp proteins. The importance of protein flexibility in the accommodation of multiple proteins around a single PCNA was also investigated. Finally, the physical coordination of one of these key maturation enzymes – PolB1 – with other replisome proteins was examined. It was demonstrated that PolB1 exists in a trimeric complex in vivo with two previously unidentified factors, raising the possibility of uncharacterised activities and interactions for this crucial enzyme. Taken together, these data provide new insights into functionally important protein-protein interactions within the archaeal replisome, and facilitate a greater understanding of the DNA replication machinery in both archaea and eukaryotes.
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