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DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...
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Related Experiment Video

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Author Spotlight: Investigating the Motion Dynamics of the Eukaryotic Replisome Components at the Single-Molecule Level
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Switch-like control of helicase processivity by single-stranded DNA binding protein.

Barbara Stekas1, Steve Yeo2, Alice Troitskaia2

  • 1Department of Physics, University of Illinois, Urbana-Champaign, Urbana, United States.

Elife
|March 19, 2021
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Summary

Single XPD helicase activity is regulated by interactions with RPA2, revealing a

Keywords:
dna repairhelicasemolecular biophysicsoptical tweezerssingle moleculesingle-stranded DNA binding proteinstructural biology

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Area of Science:

  • Molecular biology
  • Biochemistry
  • Structural biology

Background:

  • Helicases unwind nucleic acid duplexes using NTP hydrolysis.
  • Cellular helicase function is modulated by associated proteins, but mechanisms are unclear.
  • Superfamily 2B helicases, like XPD, are crucial in DNA repair and replication.

Purpose of the Study:

  • To elucidate the regulatory mechanism of XPD helicase activity by RPA2.
  • To investigate how accessory proteins influence helicase processivity.
  • To understand the molecular basis of helicase regulation by protein-protein interactions.

Main Methods:

  • Utilized optical trap measurements to monitor single XPD helicase unwinding activity.
  • Investigated the effect of replication protein A 2 (RPA2) on XPD translocation and unwinding.
  • Introduced a point mutation at a regulatory DNA binding site on XPD to assess its impact.

Main Results:

  • XPD helicase exists in two states with differing processivities.
  • Transient interactions with RPA2 stabilize the highly processive state of XPD.
  • A mutation at an XPD DNA binding site mimicked RPA2's stabilizing effect, activating a 'processivity switch'.
  • These findings reveal a novel mechanism for helicase regulation by accessory proteins.

Conclusions:

  • RPA2 acts as a regulator, stabilizing a more processive XPD state via transient interactions.
  • XPD possesses a latent 'processivity switch' activated by RPA2 or specific mutations.
  • This study provides critical insights into how accessory proteins modulate helicase function at a molecular level.