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Related Experiment Video

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Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

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Sequence-dependent base pair stepping dynamics in XPD helicase unwinding.

Zhi Qi¹, Robert A Pugh, Maria Spies

¹Center for Biophysics and Computational Biology , University of Illinois at Urbana-Champaign , Urbana , United States.

|June 7, 2013

Summary

This study reveals how XPD helicase unwinds DNA, moving one base pair at a time. Evidence suggests DNA duplex opening drives this essential step in DNA repair and transcription.

Keywords:

DNA repair None Xeroderma pigmentosum group D helicase helicase molecular motor optical tweezers single molecule

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Area of Science:

Molecular Biology
Biophysics
Biochemistry

Background:

Helicases are crucial enzymes that use ATP hydrolysis to unwind DNA duplexes.
Understanding the precise mechanism of DNA unwinding at a molecular level is essential for comprehending DNA repair and transcription processes.
High-resolution techniques are needed to directly observe helicase activity and base pair separation.

Purpose of the Study:

To investigate the DNA unwinding mechanism of the XPD helicase (a Superfamily 2 helicase) with single base pair resolution.
To elucidate the role of spontaneous duplex opening in helicase-mediated DNA translocation.
To provide direct evidence for the stepping dynamics and sequence dependence of helicase activity.

Main Methods:

Utilized optical tweezers with single base pair resolution to monitor DNA unwinding by XPD helicase.
Analyzed the stepping dynamics, including forward and backward movements, of the helicase.
Quantified the sequence-specific effects on XPD stepping behavior.

Main Results:

Demonstrated that monomeric XPD helicase unwinds duplex DNA in discrete 1-bp steps.
Observed frequent backsteps and larger conformational transitions (5-bp steps) during unwinding.
Provided strong evidence that spontaneous DNA duplex opening is utilized for forward, single-base pair stepping.

Conclusions:

The findings suggest a mechanism where spontaneous duplex opening facilitates the forward translocation of XPD helicase.
This 1-bp stepping mechanism, driven by duplex opening, may be a conserved feature among DNA helicases moving along the DNA backbone.
The study offers the most direct evidence to date for the physical process underlying helicase translocation.