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<p>DNA replication is a fundamental process that must be precisely regulated to ensure timely and faithful transmission of genetic material for proliferation of all organisms. Replication initiation is regulated through a series of precisely timed protein–DNA and protein–protein interactions. In <em>Escherichia coli</em>, one regulatory mechanism of replication initiation occurs through SeqA binding to specific sequences within the <em>oriC</em>, resulting in origin sequestration. SeqA also plays a role in chromosome organization at the replication forks. Despite the functional importance of SeqA in <em>E. coli</em>, its DNA binding mechanism has remained elusive. The work described in this thesis has shown for the first time the minimal functional unit of SeqA that forms a high-affinity complex with DNA through the loss of symmetry. This is a novel observation that explains how SeqA can distinguish template versus newly replicated strand of DNA. We have also identified a protein–protein interaction surface that separates the roles of SeqA at the origin in sequestration and at the replication forks in chromosome organization. The final contribution of the thesis is in the exploration of SeqA functions in other bacterial species and demonstrating the structural and functional similarities between <em>Vibrio cholerae </em>SeqA and <em>E. coli </em>SeqA. Together our work has made a crucial connection between the structural organization of the protein and its functional ability to bind DNA.</p> / Doctor of Philosophy (PhD)

All living organisms must organize their genome so that it not only fits within the cell, but remains accessible for cellular processes. In bacteria, an arsenal of nucleoid-associated proteins contributes to chromosome condensation. A novel nucleoid-associated protein was recently discovered in actinobacteria, and is essential in Mycobacterium. It was classified as an integration host factor protein (IHF); however, it does not share sequence or structural homology with the well characterized Escherichia coli IHF. In this study, we characterize the structure and function of Streptomyces coelicolor IHF (sIHF). We have used a combination of biochemistry and structural biology to characterize the role of sIHF in DNA binding and DNA topology. We have solved crystal structures of sIHF bound to various double-stranded DNA substrates, and show that sIHF is able to contact DNA at multiple surfaces. Furthermore, sIHF inhibits the activity of TopA, impacting DNA topology in vitro. Our work demonstrates that sIHF is a novel nucleoid-associated protein with key roles in condensing DNA. We believe that sIHF performs its function by differentially using multiple nucleic-acid binding surfaces. Further characterization is required to confirm this hypothesis in vivo. Given that the Mycobacterium homolog of sIHF (mIHF) is essential, our studies lay the foundation to explore novel drug targets for Mycobacterium tuberculosis and Mycobacterium leprae. / Thesis / Master of Science (MSc) / Unconstrained, the bacterial genome exceeds the size of the cell by 1 000- 10 000 times; thus, compacting it into a condensed structure, known as the nucleoid, is essential for life. An arsenal of nucleoid-associated proteins contributes to this process. In this study, we characterize the structure and function of a novel nucleoid–associated protein from the soil dwelling organism Streptomyces coelicolor. We used a combination of genetics, biochemistry, and structural biology to characterize the role of this protein in DNA binding and nucleoid organization. Since this protein is also found in important human pathogens, this work lays the foundation to explore the use of nucleoid-associated proteins as antimicrobial drug targets.

Quorum sensing in Gram-negative bacteria is best understood in the bioluminescent marine microorganism, <i>Vibrio fischeri</i>. In <i>V. fischeri</i>, the luminescence or <i>lux</i> genes are regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule (3-oxo-hexanoyl homoserine lactone). LuxR, which binds to the <i>lux</i> operon promoter at position -42.5, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the <font face = "symbol">a</font>CTD of RNAP in LuxR-dependent transcriptional activation of the <i>lux</i> operon promoter has been investigated. The effect of seventy alanine substitution variants of the <font face = "symbol">a</font> subunit was determined <i>in vivo</i> by measuring the rate of transcription of the <i>lux</i> operon via luciferase assays in recombinant <i>Escherichia coli</i>. The mutant RNAPs from strains exhibiting at least two fold increased or decreased activity in comparison to the wild-type were further examined by <i>in vitro</i> assays. Since full-length LuxR has not been purified to date, an autoinducer-independent N-terminal truncated form of LuxR, LuxR<font face = "symbol">D</font>N, was used for <i>in vitro</i> studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxR<font face = "symbol">D</font>N, and fourteen residues in the <font face = "symbol">a</font>CTD were identified as having negative effects on the rate of transcription from the <i>lux</i> operon promoter. Five of these fourteen residues were also involved in the mechanism of both LuxR and LuxR<font face = "symbol">D</font>N-dependent activation <i>in vivo</i> and were chosen for further analysis by DNA mobility shift assays. Results from these assays indicate that while the wild-type <font face = "symbol">a</font>CTD is capable of interacting with the <i>lux</i> DNA fragment tested, all five of the variant forms of the <font face = "symbol">a</font>CTD tested appear to be deficient in their ability to recognize and bind the DNA. These findings suggest that <font face = "symbol">a</font>CTD-DNA interactions may play a role in LuxR-dependent transcriptional activation of the <i>lux</i> operon during quorum sensing. / Master of Science

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.

No / The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a class of DNA minor groove binding agents that react covalently with guanine bases, preferably at Pu-G-Pu sites. A series of three fluorescent PBD¿coumarin conjugates with different linker architectures has been synthesized to probe correlations between DNA binding affinity, cellular localization and cytotoxicity. The results show that the linker structure plays a critical role for all three parameters. Graphical abstract A series of three fluorescent PBD¿coumarin conjugates with different linker architectures has been synthesized to probe correlations between DNA-binding affinity, cellular localization and cytotoxicity.