SINGLE-MOLECULE FLUORESCENCE STUDIES OF DNA BENDING DURING PROKARYOTIC MISMATCH REPAIR INITIATION

DNA mismatch repair (MMR) is a process that is responsible for repairing base-base mismatches and insertion/deletion loop errors incorporated during DNA replication. In humans, deficiencies in MMR are linked to cancers, including Lynch Syndrome. MMR is initiated by MutS in prokaryotes (or the MutS homologs in eukaryotes), which is responsible for recognizing the error in the DNA. Upon error recognition, MutS undergoes ATP-dependent conformational changes to form a sliding clamp state that moves along the length of the DNA. This state is thought to be important for downstream repair events, such as recruitment of the second protein in the pathway, MutL. Recent single-molecule fluorescence studies have led to a refined model for error recognition and sliding clamp formation by Thermus aquaticus MutS. While it is well established that MutS bends DNA and that this DNA bending is dynamic in the absence of nucleotides, little evidence for the DNA bending status through MMR initiation exists in the presence of ATP. In this work, the nucleotide dependence of Thermus aquaticus MutS-induced DNA bending throughout sliding clamp formation is characterized. The current model for MutS conformational changes is then modified to reflect the newfound DNA bending information. To this end, single-molecule fluorescence resonance energy transfer (smFRET) between two arms of specially designed DNA oligonucleotides is monitored. These substrates are designed such that changes in DNA bending would result in changes in FRET efficiency. Finally, a data analysis pipeline developed specifically for high throughput analysis of data of this type is presented.Doctor of Philosoph

Development and Synthesis of Utrophin Actin Binding Domain 1 (ABD1)

University Honors Capstone Project and Poster, University of Minnesota Duluth, 2016. Kate McMahon authored paper and poster; Ben Horn, Dr. Jacob Gauer, and Dr. Anne Hinderliter authored poster.Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disease containing point mutations in the muscle protein Dystrophin causing the protein to lose its function. Specifically, Dystrophin is critical for dissipating the mechanical stress placed on muscles during physical activity. Although Dystrophin is nonfunctional in DMD patients, its fetal homolog, Utrophin, is often present in higher amounts than common to adult cells. Because Utrophin and Dystrophin share 85% homology in their first actin binding domains (ABD1), the interrelatedness of structure and function validate Utrophin as a proposed therapeutic tool for combating DMD. To test this hypothesis, the thermodynamic character of Utrophin ABD1 and Dystrophin ABD1 will be compared. As Utrophin is not regularly studied, the gene for Utrophin ABD1 was designed, synthesized, and expressed in E.coli cells. Prokaryotic cells were utilized to express a eukaryotic protein because of rapid growth rate and the presence of an extra, self-replicating, circular DNA called a plasmid. A plasmid is evolutionarily advantageous because it can be passed quickly from prokaryotic cell to prokaryotic cell without the entire genome replicating, thus increasing variability. This unique attribute was utilized to express Utrophin ABD1 in E. coli cells. Although eukaryotic systems often have posttranslational modifications, this did not pose a threat for the prokaryotic cell amplification. The gene encoding the protein was designed using specific amino acid residues, not nucleotide sequences; the splicing of nucleotide sequences was irrelevant as posttranslational modification occurs before the amino acids are assembled into their primary structure. Specifically, Utrophin ABD1 was designed with BamHI and XhoI restriction enzymes flanking the 246 amino acid Utrophin ABD1 construct which was synthesized in a pUC57 E. coli plasmid. Using BamHI and XhoI, the amino acid sequence was restriction digested and subcloned into an expression vector containing components critical for nickel column chromatography like a histidine tag, TEV protease cut site, and maltose binding protein. The expression vector also contains a selective marker to find the correct ligated species such as the antibiotic Kanamycin. These plasmids were transformed into competent E. coli cells so the E. coli cells would replicate the inserted DNA the same way it replicates a plasmid. During the rapid growth, inclusion bodies, protein aggregates of overexpressed protein, are accounted for by the addition of the maltose binding protein which maintains solubility. The transformed cells were stored in a glycerol stock. Synthesis of this gene then allows growth and purification of the Utrophin ABD1 protein in a similar manner to those already classified for histidine tagged proteins. Purification is carried out at a pH of 8 so that the six histidines will be deprotonated and bind to the nickel column, thus washing out all other protein expect for the Utrophin bound to the column. Purification is important in that it insures pure protein by cleaving off the maltose binding protein using the tobacco etch virus (TEV) protease that recognizes a specific nucleotide sequence rarely found in the eukaryotic genome. Finally, thermodynamic analysis of this protein will give insight into the structure and function of Utrophin ABD1 and its potential capabilities as a therapeutic agent for patients with DMD

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca2+ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca2+ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca2+ affinity to a membrane-associated, higher Ca2+ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca2+ influx is the basis for the cooperative response of annexin a5 toward Ca2+, and the role of membrane organization in this response

Transcription errors induce proteotoxic stress and shorten cellular lifespan

Transcription errors occur in all living cells; however, it is unknown how these errors affect cellular health. To answer this question, we monitored yeast cells that were genetically engineered to display error-prone transcription. We discovered that these cells suffer from a profound loss in proteostasis, which sensitizes them to the expression of genes that are associated with protein-folding diseases in humans; thus, transcription errors represent a new molecular mechanism by which cells can acquire disease. We further found that the error rate of transcription increases as cells age, suggesting that transcription errors affect proteostasis particularly in aging cells. Accordingly, transcription errors accelerate the aggregation of a peptide that is implicated in Alzheimer’s disease, and shorten the lifespan of cells. These experiments reveal a novel, basic biological process that directly affects cellular health and aging

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca2+) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca2+ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca2+ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca2+ affinity to a membrane-associated, higher Ca2+ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca2+ influx is the basis for the cooperative response of annexin a5 toward Ca2+, and the role of membrane organization in this response

Numerical Modelling of Cryogenic Two-phase Flow in the DLR TAU Code

The present paper discusses work that has been conducted to extent the German Aerospace Center (DLR) TAU Code to simulate heat and mass transfer in two-phase systems in launcher cryogenic upper stages. Preceding work has been performed to extend the incompressible version of the DLR inhouse CFD Code TAU to simulate isothermal twophase flows. A Volume of Fluid (VOF) approach and a finite volume discretization are used. Due to the grid handling in TAU an algebraic solution of the interface position is necessary yielding shorter computation times compared to geometrical approaches on both structured and unstructured grids. This model is currently extended to simulate heat and mass transfer. Therefore, the energy conservation equation needs to be included. Density deviations are included via the Boussinesq-approximation and heat convection is modeled by Fouriers law. Basic test cases like laminar flow in a pipe were used to verify the proper functionality of the new implementation and show good agreements with analytically solutions