Author ORCID Identifier
https://orcid.org/0000-0003-4169-6342
Date of Award
5-31-2026
Document Type
Campus Access Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Dr. Daniel Dowling
Abstract
Proteins capable of binding DNA and enabling chemical transformations have played a significant role in gene expression and editing technologies. Fundamental to these activities are the evolved tools for binding DNA with affinity and potential specificity. Transcription factors and repressors must bind DNA sequences with both specificity and potent affinity; however, enzyme systems that function to alter DNA may only require affinity depending on the reaction catalyzed. This dissertation explores the human capicua transcriptional repressor and the bacteriophage 5-hydroxymethyl deoxyuracil (5hmdU) demodification enzyme.
Capicua is a transcription regulator that binds to an octameric DNA sequence found in polyomavirus enhancer activator 3 (PEA) genes, ETV1, 4, and 5, which regulate downstream processes such as cell cycle progression, cell proliferation, and migration. Capicua deficiencies also play a part in specific disease progression such as gliomagenesis and human lung adenocarcinoma. Capicua binds to an octameric TGAATGAA sequence within DNA and inducing a bend in the DNA strand. It has been observed that capicua requires at least two DNA-binding domains, HMG-box and C1, to bind to consensus DNA and induce repression of transcription. However, the specific interactions between these two domains and DNA had yet to be elucidated and how the effects of specific mutations within these domains relate to their role in cancer had yet to be studied. Additionally, the specific role of the C1 domain in binding of DNA was not yet known. Difficulties in determining the structure of this protein arise due to a large, disordered region, over one thousand amino acids long, between the two DNA-binding domains. To address this, a minimal-linker construct was designed containing the two binding domains with 18 amino acid residues between them. We report the 2.95 Å resolution crystal structure of the capicua construct, bound to a DNA oligomer containing the octameric sequence. Both the HMG-box and C1 domains are presented as tri-helical DNA-binding domains bound along the minor groove of the oligomer and inducing a 66° bend. The C1 domain, in contrast, binds to the opposite side of the TGAATGAA consensus sequence in the major groove, where it makes multiple hydrogen-bonding interactions with the backbone of the DNA. The crystal structure provides the first structural details into how the HMG-box and C1 domain together provide binding affinity and specificity for the TGAATGAA sequence.
Bacteriophages are viruses that infect target bacterial hosts and represent an important area of exploration for treatments of nosocomial antibiotic-resistant infections, with some of these infections being completely resistant to our last line of defense antibiotics. Alternatives to traditional antibiotics are being explored to address these antibiotic-resistant bacterial infections, and bacteriophages may offer one route of treatment. Previous research has shown that bacteriophages can incorporate DNA modifications, such as 5-hydroxymethyl uracil (5hmdU), in place of thymidine to evade the restriction enzyme defenses of their hosts. Additionally, some phages have shown the ability to further hypermodify 5hmdU, providing protection against different restriction enzymes. However, the mechanisms of hypermodification have yet to be elucidated.
Pseudomonas aeruginosa, of the commonly antibiotic-resistant class of bacteria, can be infected by the phage M6 which has been observed to incorporate a 5hmdU hypermodification. The M6 phage infects laboratory strains of P. aeruginosa, which is an antibiotic-resistant class of bacteria found in nosocomial infections. Therefore, the M6 phage represents an important model system. This phage encodes genes that incorporate the modified base 5hmdU and either hypermodify 5hmdU utilizing a cluster of phage-encoded enzymes or revert the modified base back to a canonical thymidine. To elucidate the function of the demodifying enzyme, we report the 2.11 Å resolution crystal structure of M6 gp55. The structure of M6 gp55 is comprised of two bundles of helices with an electropositive interface expected to interact with DNA. Based on the crystal structure, the 5hmdU modified nucleobase is predicted to flip into the active site and undergo demodification by nucleophilic attack of a conserved cysteine, resulting in the loss of water at the C5 hydroxymethyl position and production of an exocyclic methylene. Data from collaborators has revealed that both NADH and NADPH can be utilized to reduce this exocyclic methylene, resulting in the release of the nucleobase as a demodified thymidine. Additionally, mutational studies of active site residues and a disordered loop on the interface of the enzyme indicate the importance of key conserved residues in 5hmdU demodification. This research provides structural insight into hypermodification enzymes in phage to allow for future bioengineering of DNA tools and potential phage therapies.
Recommended Citation
Webb, Jonathan, "Explorations of protein-DNA binding and nucleobase hypermodification: A study of cancer mutations in the human Capicua transcriptional regulator and bacteriophage pyrimidine hypermodification and demodification enzymes" (2026). Graduate Doctoral Dissertations. 1163.
https://scholarworks.umb.edu/doctoral_dissertations/1163
Comments
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