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.

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.

No description available.

T-cell Intracellular Antigen-1 (TIA1) is a multi-domain RNA-binding protein involved in stress granule formation and implicated in neurodegenerative diseases. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids, and a C-terminal intrinsically disordered prion-related domain (PRD), which plays a role in promoting liquid-liquid phase separation. Motivated by our previous findings indicating that RRMs 2 and 3 exhibit a well-ordered structure in the oligomeric full-length form, whereas RRM1 and PRD demonstrate a propensity for phase separation, the present work in this dissertation aims to investigate the functional competence of the oligomeric state and its binding capabilities. Moreover, the study explores the effects of ligand binding on oligomerization dynamics and potential alterations in protein conformation primarily using solid-state NMR methods. The NMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at RRM2, but also weakly at RRM3, and Zn2+ binds primarily to RRM3. The binding of Zn2+ and DNA was reversible and without the formation of amyloid fibrils. The addition of Zn2+ caused the TIA1:DNA complexes to collapse, indicating that Zn2+ may play a regulatory role by shifting the nucleic acid binding off RRM3 and onto RRM2 by occupying various “half” binding sites on RRM3 and introducing a mesh of crosslinks in the supramolecular complex. Furthermore, this dissertation presents an investigation into the interdomain interactions between RRM2 and RRM3, facilitated by the successful preparation of segmentally labeled protein samples using the trans-splicing approach. The results confirm the hypothesis that Zn2+ can bring RRM2 and RRM3 closer together by crosslinking different monomers, as evidenced by the observation of enhanced NMR signals from heteronuclear correlations around the Zn2+ binding sites. In conclusion, studying the structure of full-length TIA1 oligomers is expected to reveal the mechanisms by which an RNA regulatory protein assembles and binds to its biologically relevant ligands while preserving a highly ordered oligomeric structure.

On TFBS motif discovery, three novel GA based algorithms are developed, namely GALF-P with focus on optimization, GALF-G for modeling, and GASMEN for spaced motifs. Novel memetic operators are introduced, namely local filtering and probabilistic refinement, to significantly improve effectiveness (e.g. 73% better than MEME) and efficiency (e.g. 4.49 times speedup) in search. The GA based algorithms have been extensively tested on comprehensive synthetic, real and benchmark datasets, and shown outstanding performances compared with state-of-the-art approaches. Our algorithms also "evolve" to handle more and more relaxed cases, namely from fixed motif widths to most flexible widths, from single motifs to multiple motifs with overlapping control, from stringent motif instance assumption to very relaxed ones, and from contiguous motifs to generic spaced motifs with arbitrary spacers. / TF-TFBS associated sequence pattern (rule) discovery is further investigated for better deciphering protein-DNA interactions in regulation. We for the first time generalize previous exact TF-TFBS rules to approximate ones using a progressive approach. A customized algorithm is developed, outperforming MEME by over 73%. The approximate TF-TFBS rules, compared with the exact ones, have significantly more verified rules and better verification ratios. Detailed analysis on PDB cases and conservation verification on NCBI protein records illustrate that the approximate rules reveal the flexible and specific protein-DNA interactions with much greater generalized capability. / The comprehensive pattern discovery algorithms developed will be further verified, improved and extended to further deciphering transcriptionial regulation, such as inferring whole gene regulatory networks by applying TFBS and TF-TFBS patterns discovered and incorporating expression data. / Transcription Factor (TF) and Transcription Factor Binding Site (TFBS) bindings are fundamental protein-DNA interactions in transcriptional regulation. TFs and TFBSs are conserved to form patterns (motifs) due to their important roles for controlling gene expressions and finally affecting functions and appearances. Pattern discovery is thus important for deciphering gene regulation, which has tremendous impacts on the understanding of life, bio-engineering and therapeutic applications. This thesis contributes to pattern discovery involving TFBS motifs and TF-TFBS associated sequence patterns based on Evolutionary Computation (EC), especially Genetic Algorithms (GAs), which are promising for bioinformatics problems with huge and noisy search space. / Chan, Tak Ming. / Advisers: Kwong-Sak Leung; Kin-Hong Lee. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 147-153). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.

To maintain the genomic integrity, cell has evolved various DNA repair pathways. Base Excision Repair pathway (BER) is one such DNA repair pathway which is dedicated to protect DNA from small lesions such as oxidation, alkylation, deamination and loss of bases. Uracil is a promutagenic base which appears in the genome as a result of misincorporation of dUTP or due to oxidative deamination of cytosine. Uracil-DNA glycosylases (UDGs) are DNA repair enzymes that initiate multistep base excision repair (BER) pathway to excise uracil from DNA. Excision of uracil generates an abasic site (APDNA). AP-sites are cytotoxic and mutagenic to the cell. AP endonucleases act downstream to UDG in this pathway and generate substrates for DNA polymerase to fill in the correct bases. The cytotoxicity of AP-sites raises the question whether uracil excision activity is coupled to AP endonuclease activity. Also, there is transient formation of single stranded DNA (ssDNA) during DNA metabolic processes such as replication, repair and recombination. ssDNA is more prone to various nucleases and DNA damaging agents. All the living organisms encode single stranded DNA binding protein (SSB) that binds to ssDNA and protects it from various damages. In addition, SSB plays a vital role during DNA replication, repair and recombination. Studies on SSBs from prototype Escherichia coli and an important human pathogen, Mycobacterium tuberculosis have shown that despite significant variations in their quaternary structures, the DNA binding and oligomerization properties of the two are similar. My PhD thesis consists of four Chapters. Chapter 1 summarizes the relevant literature review on DNA damage and repair with an emphasis on uracil DNA glycosylase and its interacting protein, SSB. Chapters 2 and 3 describe my studies on the mechanism of uracil excision repair in E. coli. Chapter 4 describes my findings on the structure-function relationship of single stranded DNA binding proteins from E. coli and M. tuberculosis. Specific details of my research are summarized as follows: (1) Analysis of the impact of allelic exchange of ung with a mutant gene encoding Uracil DNA Glycosylase attenuated in AP-DNA binding in the maintenance of genomic integrity in Escherichia coli. There are five families of UDGs. Of these, Ung proteins (family 1 UDGs) represent highly efficient and evolutionary conserved enzymes. Structural and biochemical analysis of Ung proteins has identified two conserved motif, motif A (62GQDPY66) and motif B (187HPSPLS192) in E. coli that are important for the catalysis by Ung enzyme. Y66 of motif A is in van der Waals contact with the C5 position of the uracil and prevents entry of other bases. Earlier study from the laboratory showed that the Y66W and Y66H mutants of Ung were compromised by ~7 and ~170 fold, respectively in their uracil excision activities. However, unlike the wild-type and Y66H proteins, Y66W was not inhibited by its product (uracil or AP-DNA). In this study, by fluorescence anisotropy measurements I have shown that compared with the wild-type protein, the Y66W mutant is moderately compromised and attenuated in binding to AP-DNA. Allelic exchange of ung in E. coli with ung::kan, ungY66H:amp or ungY66W:amp alleles showed ~5, ~3.0 and ~2.0 fold, respectively increase in mutation frequencies. Analysis of mutations in the rifampicin resistance determining region (RRDR) of rpoB revealed that the Y66W allele resulted in an increase in A to G (or T to C) mutations. However, the increase in A to G mutations was mitigated upon expression of wild-type Ung from a plasmid borne gene. Biochemical and computational analyses showed that the Y66W mutant maintains strict specificity for uracil excision from DNA. Interestingly, a strain deficient in AP-endonucleases also showed an increase in A to G mutations. These findings have been discussed in the context of a proposal that the residency of DNA glycosylase(s) onto the AP-sites they generate shields them until recruitment of AP-endonucleases for further repair. It is proposed that an error prone replication against AP-sites (as a result of uracil excision activities on A:U pair) may result in A to G mutations. 2. Mechanism of appearance of A to G mutations in ungY66W:amp strain of Escherichia coli. In this part of my study, I have investigated the role of error prone DNA polymerases in the mutational specificity of ungY66W:amp strain. It was observed from various studies in E. coli that, DNA polymerase IV (Pol IV) and DNA polymerase V (Pol V) are involved in error-prone replication on damaged or AP-site containing DNA. E. coli strains containing deletion of either dinB (encoding DNA Pol IV) or umuDC (encoding DNA Pol V) were generated and used to study mutation frequency and mutation spectrum. Deletion of DNA Pol V resulted in a decrease in A to G mutations in ungY66W:amp E. coli strain, suggesting that increase in A to G mutations were a consequence of error prone incorporation by DNA Pol V. 3. Structure and Function studies on Single Stranded DNA Binding Proteins from Escherichia coli and Mycobacterium tuberculosis. SSB from M. tuberculosis (MtuSSB) has similar domain organization as the EcoSSB. Moreover, the biochemical properties such as oligomerization, DNA binding affinity and minimum binding site size requirements were shown to be similar to EcoSSB. However, structural studies suggested that quaternary structures of these two SSBs are variable. In this study I have used X-ray crystal structure information of these two SSBs to generate various chimeras after swapping at various regions of SSBs. Chimeras mβ1, mβ1’β2, mβ1-β5, mβ1-β6, and mβ4-β5 SSBs were generated by substituting β1 (residues 611), β1’β2 (residues 21-45), β1-β5 (residues 1 to 111), β1-β6 including a downstream sequence (residues 1 to 130), and β4-β5 (residues 74-111) regions of EcoSSB with the corresponding sequences of MtuSSB, respectively. Additionally, mβ1’β2ESWR SSB was generated by mutating the MtuSSB specific ‘PRIY’ sequence in the β2 strand of mβ1’β2 SSB to EcoSSB specific ‘ESWR’ sequence. Biochemical characterization revealed that except for mβ1 SSB, all chimeras and a control construct lacking the C-terminal domain (ΔC SSB) efficiently bound DNA in modes corresponding to limited and unlimited modes of binding. The mβ1 SSB was also hypersensitive to chymotrypsin treatment. The mβ1-β6, MtuSSB, mβ1’β2 and mβ1-β5 constructs complemented E. coli Δssb in a dose dependent manner. Complementation by the mβ1-β5 SSB was poor. In contrast, mβ1’β2ESWR SSB complemented E. coli as well as EcoSSB. Interestingly, the inefficiently functioning SSBs resulted in an elongated cell/filamentation phenotype of E. coli. Taken together, our observations suggest that specific interactions within the DNA binding domain of the homotetrameric SSBs are crucial for their biological function.