Date of Award

7-31-2023

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Developmental, Regenerative, & Stem Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Abstract Mycobacterium tuberculosis (Mtb) causes tuberculosis disease in humans and is one of the leading causes of death worldwide due to infectious agents. During infection, Mtb is exposed to reactive oxygen species and reactive nitrogen intermediates from the host immune response that causes DNA damage. UvrD-like helicases are involved in DNA repair and use energy from ATP hydrolysis to translocate on single stranded DNA (ssDNA) or unwind double-stranded DNA (dsDNA) to remove the damaged DNA strand. Previous studies on UvrD-like helicases have shown that they exist in a monomer-dimer equilibrium and unwind only as dimers in the absence of accessory factors. However, Mtb UvrD1 had previously been suggested to perform unwinding as a monomer. Using quantitative biophysical assays including analytical ultracentrifugation and stopped-flow kinetics, I demonstrated that Mtb UvrD1 exists in monomer, dimer, and higher-order oligomeric forms, where the dimer is redox-dependent. I identified a cysteine in the 2B domain that is required for oxidative dimerization, thus demonstrating that the 2B domain is directly involved in dimerization. Furthermore, although the monomer of UvrD1 can bind and translocate on ssDNA, only the dimer unwinds dsDNA supporting the notion that helicase activity requires more than just translocation on ssDNA. My results suggest a model where UvrD1 senses the oxidative conditions within human macrophages during infection through dimerization, resulting in the activation of its DNA-unwinding activity needed for DNA repair and other DNA metabolic pathways. In addition, it also highlights the dimerization interface of the UvrD-family helicases that was previously unknown. In related work using cryo-electron microscopy, I have visualized the structure of the Mtb UvrD1 dimer where the disulfide bond is clearly visible. Data analysis resulted in 4-5 Å resolution structures of UvrD1 in two different conformations (open and closed) that might represent two different conformations of a dimer. Independent studies have shown that the helicase activity of Mtb UvrD1 is activated by Mtb Ku which is a homodimer homologous to the eukaryotic Ku70/Ku80 heterodimer and which plays a role in double-stranded break repair via non-homologous end-joining. I followed up on this observation in the context of my results of the redox-sensitive dimeric structure of UvrD1 and showed that Ku specifically activates the unwinding activity of a monomer of UvrD1. Importantly, this activity is weak compared to that of the dimer and is only observed under multiround conditions and therefore depends on multiple interactions between UvrD1 monomer, Ku, and DNA. Dissecting the interaction further, I revealed that the Ku-UvrD1 interaction occurs via the C-terminal Tudor domain of UvrD1. I also found that 3 Ku dimers are bound to the DNA substrate and that Ku appears to be able to load on dsDNA even in the presence of ssDNA overhangs. Since the maximal Ku-dependent unwinding occurs under saturating conditions of Ku, my results suggest a model where more Ku dimers bound to DNA results in more unwinding. In summary, my characterization of Mtb UvrD1 highlights its potential role in multiple DNA repair pathways through different mechanisms of activation and contributes to our understanding of the UvrD-family of helicases which are found across biology.

Language

English (en)

Chair and Committee

Eric Galburt

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Included in

Biochemistry Commons

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