Unveiling Dna Polymerase Synthesis And Proofreading Activities - One Molecule At A Time

DNA polymerases maintain the genome integrity from one generation to the next by faithfully synthesizing new DNA and by participating in DNA repair processes. The Klenow fragment of E. coli DNA polymerase I has served as a model polymerase for decades because it is straightforward to purify, well-characterized kinetically, and it is able to carry out DNA synthesis and proofreading. In 2009, Christian, et al. developed a single molecule Förster resonance energy transfer (smFRET) approach to monitor the polymerase position on the DNA with single base pair resolution. We have worked to optimize nearly every aspect of that approach and to apply the technique to characterize DNA polymerase dynamics on the DNA with unprecedented detail. First, we improved the cysteine-labeling protocol to ensure the labeling was site-specific and efficient. Second, we applied a new single molecule fluorescence technique, single molecule protein induced fluorescence enhancement (smPIFE), to our system to accurately measure binding dynamics and to strengthen our interpretation of the polymerase position on the DNA. Third, determined how carcinogenic DNA adducts disrupt polymerase binding on the DNA by using smFRET and smPIFE. Accordingly, we designed the experiment to distinguish between pol and exo site binding in real-time, and we identified a novel intermediate state in the proofreading mechanism. Finally, we tracked individual polymerases as they incorporated bases on the DNA. These experiments support the existence of a fidelity-checking step following each incorporation. The assays and applications described herein lay the groundwork for future DNA polymerase mechanistic studies

Single-molecule microscopy reveals new insights into nucleotide selection by DNA polymerase I.

The mechanism by which DNA polymerases achieve their extraordinary accuracy has been intensely studied because of the linkage between this process and mutagenesis and carcinogenesis. Here, we have used single-molecule fluorescence microscopy to study the process of nucleotide selection and exonuclease action. Our results show that the binding of Escherichia coli DNA polymerase I (Klenow fragment) to a primer-template is stabilized by the presence of the next correct dNTP, even in the presence of a large excess of the other dNTPs and rNTPs. These results are consistent with a model where nucleotide selection occurs in the open complex prior to the formation of a closed ternary complex. Our assay can also distinguish between primer binding to the polymerase or exonuclease domain and, contrary to ensemble-averaged studies, we find that stable exonuclease binding only occurs with a mismatched primer terminus

Master of Science

thesisThis research was an effort to create and test a performance-based mix design that allows for oil sand to be incorporated as the binder material. The design is based on creating an open-graded aggregate blend that will allow incorporating the optimum volume of oil sand. The oil sand used in this project is a natural, unmodified material that was characterized to explore its unique attributes. Aggregate stockpiles were blended to find the maximum space for the oil sand to be added in the mix. A field project was performed to bring the laboratory mix to full-size scale. Even though there were some minor delays while trying to incorporate the oil sand into the mixing chain, the results prove that the design from this work was feasible. Loose mix samples were obtained and brought back to the laboratory for testing. The Hamburg Wheel Tracking Device (HWTD), Bending Beam Rheometer (BBR), and Asphalt Mixture Performance Tester (AMPT) were performed on the loose mix samples from the field project. The results were compared with the laboratory mix results as well as with results from conventional hot mix asphalt (HMA) mixes used in Utah. The HWTD shows the mix has a solid aggregate structure; however, there is a potential for premature pavement failure due to moisture susceptibility. In the BBR, all of the oil sand mixes had a iv greater creep stiffness at low temperatures than conventional binder grades; meaning that the oil sand mixtures are more likely to crack at low in-service temperatures when compared to the performance binders typically used in interstate roads. In the AMPT, at high in-service temperatures, the dynamic modulus was lower than the conventional mixes, meaning there is a greater likelihood of rutting occurring at high temperatures because the binding material is softer. Based on these performance measures, it was determined that it is possible to develop a mixture that incorporates oil sands into a pavement material for rural or low volume roads. There are economic and environmental advantages to using oil sand mixes as a roadway material. Oil sand mixes require a lower mixing temperature than HMA and, therefore, can save on both fuel costs and emissions

Carcinogenic adducts induce distinct DNA polymerase binding orientations

DNA polymerases must accurately replicate DNA to maintain genome integrity. Carcinogenic adducts, such as 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF), covalently bind DNA bases and promote mutagenesis near the adduct site. The mechanism by which carcinogenic adducts inhibit DNA synthesis and cause mutagenesis remains unclear. Here, we measure interactions between a DNA polymerase and carcinogenic DNA adducts in real-time by single-molecule fluorescence. We find the degree to which an adduct affects polymerase binding to the DNA depends on the adduct location with respect to the primer terminus, the adduct structure and the nucleotides present in the solution. Not only do the adducts influence the polymerase dwell time on the DNA but also its binding position and orientation. Finally, we have directly observed an adduct- and mismatch-induced intermediate state, which may be an obligatory step in the DNA polymerase proofreading mechanism

Autonomous Gyroscopic 2-Wheel Differential Robot

This paper demonstrates the implementation of an autonomous gyroscopic 2-wheel differential robot, including a forward and inverse kinematics simulation in MatLAB, a test hardware robot and programmed demonstration of a simple move forward and spin left motion, and a final configuration of a complete square path based on programming kinematics, gyroscopic speed responses, and remote-control functionality

Gentleman Johnny Plays War: John Burgoyne and The Blockade of Boston

John Burgoyne, a British general during the American Revolution, is best known for his defeat by the Continental Army at Saratoga. In addition to serving as a general,Burgoyne was a playwright. While in Boston during the blockade following the Battle ofBunker Hill Burgoyne combined his interests, writing a satire of the war. The Blockade of Boston, Burgoyne\u27s play, was first presented as an afterpiece to a production of The Busybody on January 8, 1776 (Silverman 292).Accounts of the performance differ in detail, but the central event is consistent: during the performance a soldier walked out on stage and announced that the rebels were attacking a British position. The audience of British military personnel, believing the statement to be a part of the performance, stayed in their seats to enjoy the show, only to then realize their mistake a moment later and rush off in great confusion. Most of the surviving records of this event are from the view of the delighted revolutionaries, who published accounts of it in their newspapers and pamphlets, to the lasting humiliation of the men involved with the production.I first encountered the story of Burgoyne\u27s Blockade of Boston while working as a teaching assistant in an undergraduate theatre history class. The professor, Noreen Barnes, was lecturing on American theatre in the eighteenth century when she told the story of the interrupted first performance. I was intrigued by the story, and so when I wrote a paper on a disrupted performance for a historiography class, I chose to research the topic. I discovered that The Blockade of Boston, in addition to being a great story in its own right, could serve as a lens to examine the history of the period, opening questions of race, gender, and just what it means to be an American

Teaching Theatre History: Re-Directing an Existing Course

This thesis is written to detail the process and results of the theatre history class that I assisted in teaching during the fall semester of 2005. It details the process of formulating the class structure, how the class proceeded, my lecture process, and finally the responses that the students had to the class. The first chapter of this paper goes into the process of getting the class formulated. When Shaun McCracken and I were asked to take over instruction of THEA 307 for the fall during Dr. Barnes\u27 sabbatical, rather than exactly following the process that the class had previously taken, we decided to alter some of the elements of the class. We added in a response system for the reading that the students would be doing through the semester. In addition to this, we put together a course packet of readings from plays and other books and essays to supplement the main book we would be using, rather than have the students buy another expensive book for the course that they would not entirely use throughout the semester, indeed the rest of the year. For the second chapter, I go into the class itself, its environment, the physical shape of the class as it went on and the grading for the semester. Third in this thesis is a look at the lectures that I performed for my section of the class time, the rationale behind the section layout, and where some of the readings had their basis in my assignment. Finally, the paper looks at the responses that the students had to the class itself and any possible improvements that I could be made in future classes of this type

Lessons in waste minimization from nuclear industry experience

The nuclear power industry has been very successful at reducing waste volumes and waste sources. The success has been driven by escalating cost, decreasing disposal ability, and a desire by the industry to achieve excellence. The result has been a cycle of continuing improvement resulting in reduced cost. Many of the examples of Dry Active Waste reduction are applicable to the Department of Energy in both operations and remedial activities. This paper discusses several successful examples of utility applications in this area