Single-molecule optical trapping studies of the regulation mechanisms of XPD helicase

Yeo, Steve Hwansoo

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https://hdl.handle.net/2142/132642 Copy

Description

Title

Single-molecule optical trapping studies of the regulation mechanisms of XPD helicase

Author(s)

Yeo, Steve Hwansoo

Issue Date

2025-11-26

Director of Research (if dissertation) or Advisor (if thesis)

Chemla, Yann R

Doctoral Committee Chair(s)

Chemla, Yann R

Committee Member(s)

• Gruebele, Martin
• Jin, Hong
• Kim, Sangjin

Department of Study

School of Molecular & Cell Bio

Discipline

Biophysics & Quant Biology

Degree Granting Institution

University of Illinois Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

XPD, Optical tweezers

Abstract

DNA is more than just a molecule. It is the blueprint of life, encoding the instructions that connect generations, shape identities, and reveal the mysteries of biology. Maintaining DNA integrity is essential, as repair, replication, and protective networks ensure that the genetic code is faithfully preserved and transmitted, allowing organisms to thrive and pass on their legacy. Helicases, as molecular motors, play a critical role in this maintenance by unwinding DNA and RNA to enable replication, repair, and transcription. Harnessing ATP hydrolysis, helicases navigate complex nucleic acid structures, preserving genome stability and ensuring that genetic information remains accessible. Given their central role in genomic processes, understanding helicase kinetics, mechanics, and regulation is essential. By elucidating how helicases unwind nucleic acids, coordinate with other proteins, and regulate processivity, we gain insights into fundamental cellular functions, genome stability, and the molecular basis of helicase-linked diseases, opening avenues for targeted therapeutics. Advanced biophysical techniques now provide a quantitative and mechanistic approach to molecular biology, yielding insights beyond traditional observation. Among these powerful interdisciplinary tools, optical tweezers are exceptional. They allow direct, single-molecule manipulation and measurement, fundamentally advancing our understanding of essential cellular processes like molecular motor motion and protein-DNA interactions. This thesis presents studies of two helicases using optical tweezers. First, I examine the regulation mechanisms of the repair helicase Xeroderma pigmentosum group D (XPD), an iron-sulfur (Fe-S) cluster-containing helicase with medical relevance. XPD plays a critical role in DNA repair, specifically nucleotide excision repair (NER), and functions as part of the large TFIIH complex in eukaryotes. However, its mechanism in archaeal organisms, which lack many eukaryotic repair proteins, remains unclear. Using optical tweezers, I investigated how multimeric XPD from Ferroplasma acidarmanus achieves synergistic enhancement of DNA unwinding. We observed that XPD unwinds longer stretches of DNA as the loading site increases, allowing multiple XPD molecules to bind. On longer loading sites, XPD could traverse difficult-to-unwind DNA regions that monomeric XPD could not. This enhanced unwinding is achieved by preventing fork regression at these challenging positions, with longer dwell times suggesting that multiple XPDs allow repeated attempts at overcoming barriers. Interestingly, the overall average unwinding rate did not depend on the number of loaded XPDs. However, when the DNA sequence was divided into easy-to-open and hard-to-open regions, multimeric XPD exhibited synergistic increases in unwinding rate only at the easy-to-open positions. Next, I analyzed whether multimeric XPD follows a “train” or “dimer-activation” model of enhancement. The results support a train model, in which each additional XPD contributes to increased unwinding. I also attempted to correlate unwinding activity with the number of XPD molecules using fluorescently labeled XPD. While single XPD molecules produced clear fluorescent signals, multimeric XPD fluorescence behaved unexpectedly, likely due to quenching by the Fe-S cluster, indicating that further investigation and optimization are required. Replication protein A (RPA) is a ubiquitous single-stranded DNA–binding protein that protects ssDNA from damage. XPD and RPA often colocalize at DNA repair regions, and in vitro, the RPA2 subunit has been shown to enhance XPD’s DNA unwinding activity. To investigate the mechanism behind this enhancement, I examined the mutant XPDH202A, which lacks a secondary DNA interaction site, and found that, unlike wild-type XPD, it exhibited enhanced unwinding even in the absence of RPA2. While wild-type XPD shows increased unwinding in the presence of RPA2, the mechanism of this enhancement was previously unclear. My results suggest that RPA2 may disrupt inhibitory interactions at a secondary DNA-binding site, shifting XPD into a more active state. Finally, I examined the unwinding activity of the eukaryotic Drosophila melanogaster CMG helicase, a ring-forming heterohexamer that drives replication fork progression. Although previous single-molecule studies provided insights into CMG, detailed high-resolution mechanics remained unclear. Using optical tweezers, I observed two distinct types of CMG unwinding: a very slow mode, where 20 bp were unwound in over 100 s, consistent with magnetic tweezer experiments by our collaborators, and a faster mode, where 20 bp were unwound in under 50 s. The fast traces exhibited clear 4-bp periodic steps, revealing finer details of CMG dynamics.

Graduation Semester

2025-12

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/132642

Copyright and License Information

Copyright 2025 Steve Yeo

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