Single-Molecule Analysis Reveals Differential Effect of ssDNA-Binding Proteins on DNA Translocation by XPD Helicase

Masayoshi Honda, Jeehae Park, Robert A. Pugh, Taekjip Ha, Maria Spies

Research output: Contribution to journalArticle

Abstract

An encounter between a DNA-translocating enzyme and a DNA-bound protein must occur frequently in the cell, but little is known about its outcome. Here we developed a multicolor single-molecule fluorescence approach to simultaneously monitor single-stranded DNA (ssDNA) translocation by a helicase and the fate of another protein bound to the same DNA. Distance-dependent fluorescence quenching by the iron-sulfur cluster of the archaeal XPD (Rad3) helicase was used as a calibrated proximity signal. Despite the similar equilibrium DNA-binding properties, the two cognate ssDNA-binding proteins RPA1 and RPA2 differentially affected XPD translocation. RPA1 competed with XPD for ssDNA access. In contrast, RPA2 did not interfere with XPD-ssDNA binding but markedly slowed down XPD translocation. Mechanistic models of bypassing DNA-bound proteins by the Rad3 family helicases and their biological implications are discussed.

Original languageEnglish (US)
Pages (from-to)694-703
Number of pages10
JournalMolecular cell
Volume35
Issue number5
DOIs
StatePublished - Sep 11 2009
Externally publishedYes

Keywords

  • DNA
  • PROTEINS

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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