Single-molecule analysis reveals differential effect of ssDNA-binding proteins on DNA translocation by XPD helicase

Mol Cell. 2009 Sep 11;35(5):694-703. doi: 10.1016/j.molcel.2009.07.003.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / metabolism*
  • Binding Sites
  • Carbocyanines
  • DNA / chemistry
  • DNA / metabolism*
  • DNA Repair*
  • Fluorescent Dyes
  • Kinetics
  • Models, Molecular
  • Nucleic Acid Conformation
  • Protein Conformation
  • Replication Protein A / chemistry
  • Replication Protein A / metabolism*
  • Signal Processing, Computer-Assisted
  • Spectrometry, Fluorescence
  • Xeroderma Pigmentosum Group D Protein / chemistry
  • Xeroderma Pigmentosum Group D Protein / metabolism*


  • Archaeal Proteins
  • Carbocyanines
  • Fluorescent Dyes
  • Replication Protein A
  • cyanine dye 3
  • cyanine dye 5
  • Adenosine Triphosphate
  • DNA
  • Xeroderma Pigmentosum Group D Protein