No description available.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Under normal conditions, smooth muscle cells do not replicate, or proliferate, and provide a means of contraction for many internal organs, including blood vessels and the gut. However, under abnormal or disease conditions, such as congenital heart disease and cancer, smooth muscle cells acquire the ability to replicate, to make extracellular matrix proteins and to migrate. Thus, determining how smooth muscle cells regulate these processes is crucial to understanding how the cells can switch between normal and diseased states. Serum response factor (SRF) is a widely expressed protein that plays a key role in the regulation of smooth muscle differentiation, proliferation and migration. It is generally accepted that one way that SRF can distinguish between these functions is through pathway-specific co-factor interactions. A novel SRF co-factor, chromodomain helicase DNA binding protein 8 (CHD8), was originally isolated from a yeast two-hybrid assay. CHD8 is widely expressed in adult tissues including smooth muscle. Data from in vitro binding assays indicate that the N-terminus of CHD8 can interact directly with the MADS domain of SRF. Co-immunoprecipitation assays verified the ability of these two proteins to interact within cells. Adenoviral-mediated shRNA knockdown of CHD8 in smooth muscle cells resulted in statistically significant 10-20% attenuation of expression of SRF-dependent, smooth muscle-specific genes. Similar experiments revealed that knockdown of CHD8 did not affect the SRF-dependent induction of immediate early genes required to promote proliferation. In contrast, knockdown of CHD8 in A10 vascular smooth muscle cells resulted in a marked induction in of apoptosis, characterized by increases in apoptotic markers such as phospho-H2A.X, cleaved PARP and activated caspase-3. These data suggest that CHD8 may play a specific role in modulating SRF’s activity toward anti-apoptotic genes, thereby regulating smooth muscle cell survival.

Indiana University-Purdue University Indianapolis (IUPUI) / The DNA minor groove continues to be an important biological target in the development of anticancer, antiviral, and antimicrobial compounds. Among agents that target the minor groove, studies of well-established benzimidazole-based DNA binders such as Hoechst 33258 have made it clear that the benzimidazole-amidine portion of these molecules promotes an efficient, site-selective DNA association. Building on the beneficial attributes of existing benzimidazole-based DNA binding agents, a series of benzimidazole-amino acid conjugates was synthesized to investigate their DNA recognition and binding properties. In this series of compounds, the benzimidazole-amidine moiety was utilized as a core DNA “anchoring” element accompanied by different amino acids to provide structural diversity that may influence DNA binding affinity and site-selectivity. Single amino acid conjugates of benzimidazole-amidines were synthesized, as well as a series of conjugates containing 20 dipeptides with the general structure Xaa-Gly. These conjugates were synthesized through a solid-phase synthetic route building from a resin-bound amino acid (or dipeptide). The synthetic steps involved: (1) the coupling of 4-formylbenzoic acid to the resin-bound amino acid (via diisopropylcarbodiimide and hydroxybenzotriazole); followed by (2) introduction of a 3,4-diaminobenzamidoxime in the presence of 1,4-benzoquinone to construct the benzimidazole ring; and, finally, (3) reduction of the resin-bound amidoxime functionality to an amidine via treatment with 1M SnCl2•2H2O in DMF before cleavage of final product from the resin. The synthetic route developed and employed was simple and straightforward except for the final reduction that proved to be very arduous. All target compounds were obtained in good yield (based upon weight), averaging 73% mono-amino acid and 78% di-amino acid final compound upon cleavage from resin. Ultimately, the DNA binding activities of the amino acid-benzimidazole-amidine conjugates were analyzed using a fluorescent intercalator displacement (FID) assay and calf thymus DNA as a substrate. The relative DNA binding affinities of both the mono- and di-amino acid-benzimidazole-amidine conjugates were generally weaker than that of netropsin and distamycin with the dipeptide conjugates showing stronger binding affinities than the mono-amino acid conjugates. The dipeptide conjugates containing amino acids with positively charged side chains, Lys-Gly-BI-(+) and Arg-Gly-BI-(+), showed the strongest DNA binding affinities amongst all our synthesized conjugates.

No description available.

No description available.

Tn7 and Tn7-like transposons are complex elements found in disparate environments and are responsible for mobilizing a wide variety of genes and forming pathogenicity/fitness islands. They are novel in their ability to recognize both a single site in the chromosome and specifically target transposition into mobile plasmids via dedicated TnsD and TnsE targeting proteins. TnsE recognizes mobile plasmids through an association with the processivity clamp and a 3′ recessed DNA end during conjugal replication. However, the mechanism for the specific recognition of 3′ recessed DNA ends remains unclear. Structural analyses of the C-terminal domain of TnsE identified a novel protein fold including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-function variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structure-based analysis of a series of TnsE variants relates transposition to DNA binding stability. Follow up studies of full length TnsE bound to DNA are in progress. / Thesis / Master of Science (MSc)

The world of precision medicine was revolutionized by the discovery of CRISPR-Cas systems. In particular, the capabilities of the programmable nuclease Cas9 and its derivatives have unlocked a world in which applied genome engineering to cure human disease is a reality being pursued in patient clinical trials. Gene editing via the induction of programmable, site-specific double strand breaks (DSBs) has been revolutionary for the precision medicine field. However, there are many safety concerns centered on the induction of DSBs causing potential undesirable on- and off-target consequences, particularly for in vivo CRISPR applications. To circumvent these warranted concerns, many groups have attempted to repurpose recombinases or engineer new fusion systems to perform programmable genome engineering without the induction of DSBs. This dissertation will first highlight the development of recombinases for programmable DNA insertions over the course of decades, including efforts to evolve novel DNA recognition sequences, efforts to tether recombinases to programmable DNA-binding proteins, and the recent discovery of naturally occurring RNA-guided DNA transposition systems. This dissertation will then highlight the development of CRISPR-associated transposases (CASTs) as DSB-independent programmable mammalian gene editing tools capable of integrating large DNA cargos, as well as the future directions that may further enhance CAST activity in human cells. The works in this dissertation detail the initial efforts to engineer and optimize a new class of genome manipulation tools that were previously absent from the gene editing toolkit.

The conversion of genetic information into functional RNA and protein is of fundamental importance to all known life forms. In cellular organisms, this hinges on the interaction of double stranded DNA and the transcription factor class of proteins. Substantial progress in the fields of biochemistry and genomics have made the identification of transcription factor binding sites and the resultant change in transcriptional output relatively routine. However, fully understanding this central life process requires knowing not only where transcription factors bind DNA, but why and how. These questions are approached here using solution state NMR spectroscopy and the statistical technique of bootstrap aggregation in order to: i) glean biologically relevant insights into the dynamics of the GCN4 transcription factor from NMR relaxation experiments; ii) examine the influence of electrostatics on the structure of GCN4 in the absence of DNA; iii) analyze the conformational state of several Hox transcription factor DNA binding sites. NMR spectroscopy capitalizes on connections between electromagnetism and the quantum mechanical property of nuclear spin angular momentum to study the structure of molecules. Application of NMR relaxation experiments provides further information on molecular structure and dynamics. When performed in solution, the data generated by this technique occurs in conditions more similar to those found within a cell than other approaches used in structural biology. However, the biological relevance of any insights derived from solution state NMR relaxation experiments depends on the application of an appropriate model for nuclear spin relaxation. Typically, this involves applying a statistical test to select the best model from among several candidates in the model-free formalism. Chapter 3 uses 15N relaxation data collected on the basic leucine zipper (bZip) domain of the GCN4 transcription factor to detail the potential problems and model selection errors that arise from this approach, and presents the alternative method of bootstrap aggregation. Applying this statistical technique allowed for the generation of multimodel inferences about the internal motions and rigidity of the basic region of GCN4, enhancing the likelihood of their biological relevance. The results presented in Chapter 3 further confirmed the presence of nascent helices in the generally disordered basic region of the GCN4 bZip domain. Interestingly, when complexed with appropriate DNA substrate, this region assumes a fully α-helical conformation. A long standing hypothesis assumes the inability of the basic region to form an α-helix in the absence of DNA arises, in part, due to repulsion between its charged amino acids. This hypothesis is tested in Chapter 4 using NMR relaxation experiments performed in solutions containing either increased or decreased concentrations of salt. Surprisingly, screening the electrostatic repulsion between charged residues using higher levels of salt had no discernible effect on the structure or dynamics of the basic region. Chapter 5 examines the other side of the interaction between DNA and transcription factors. Here, previous work performed with the Hox family of transcription factors indicated the conformational state of DNA has an important role in enhancing the specificity with which Hox proteins bind certain sequences. In particular, the geometry of the DNA minor groove strongly influences the recruitment of appropriate Hox transcription factors. This relationship is examined using solution state NMR to study four Hox DNA binding sequences. The binding affinity between each of these sequences and the Hox protein AbdB was previously shown to correlate with the native unbound state of the DNA. The two sequences predicted to have native minor groove widths similar to those of the bound DNA had higher affinity for AbdB than those that deformed upon binding. Though mixed, the results of NMR experiments generally support the predicted structures, particularly for the high affinity sequences, indicating a single pronounced narrowing of the minor groove. Taken together, the results presented here illustrate the complex interactions underpinning the appropriate binding of DNA and transcription factors. It further highlights the need to study the structure and dynamics of both DNA and protein, as well as that of the bound complex, in order to fully understand how and why specific sequences are bound in response to stimuli.