Form and functional observations: structural mechanisms of DNA polymerase Iota and inhibition of Bacteroides Thetaiotaomicron Helicase RuvB

Chapter One

DNA replication is essential for life, yet some regions of DNA are difficult for standard enzymes to copy accurately. Human DNA polymerase Iota (Pol ι) is a specialized enzyme that helps cells overcome these obstacles. This study uses X-ray snapshots over time to see Pol ι at multiple stages of DNA synthesis, revealing how the enzyme incorporates nucleotides and stabilizes newly formed DNA. The findings show that Pol ι maintains a remarkably stable overall structure throughout the catalytic process, relying instead on subtle adjustments within its active site to perform chemistry. The enzyme consistently uses a non-traditional base-pairing strategy when copying adenine, demonstrating that this geometry is a fundamental feature of its mechanism. Key catalytic residues remain fixed, and an arginine is identified as an important contributor to nucleotide incorporation. Together, these observations provide a detailed, time-resolved view of how Pol ι enables DNA replication across challenging templates, improving our understanding of genome maintenance and enzymatic specialization.

Chapter Two

Imbalances in the gut microbiome, or dysbioses, are linked to diseases such as colorectal cancer, where bacterial biofilms promote inflammation and long-term colonization. Disrupting these persistent microbial communities without broadly eliminating beneficial bacteria remains a major therapeutic challenge. This study investigates RuvB, an essential DNA repair protein in Bacteroides thetaiotaomicron, which is a major component of the gut microbiome, as a potential target for controlling biofilm growth. Structural analyses reveal that RuvB forms a stable six-part assembly. Biochemical experiments show that a small antibody-like protein, a nanobody, binds selectively to RuvB. Although this nanobody has limited effects on free-growing bacteria, it strongly suppresses biofilm formation at low concentrations. Additional experiments confirm the specificity of this interaction and demonstrate that restoring RuvB reverses the biofilm defect. These results identify RuvB as a promising target for strategies aimed at weakening harmful biofilms and suggest that precision molecules such as nanobodies could help modulate disease-associated microbes while preserving the broader gut ecosystem.