| Title | Single-molecule analysis reveals differential effect of ssDNA-binding proteins on DNA translocation by XPD helicase. | | Author(s) | Honda M, Park J, Pugh RA, Ha T, Spies M | | Institution | Department of Biochemistry, University of Illinois at Urbana-Champaign, 61801, USA. | | Source | Mol Cell 2009 Sep 11; 35(5):694-703. | | MeSH | Adenosine Triphosphate
Archaeal Proteins
Binding Sites
Carbocyanines
DNA
DNA Repair
Fluorescent Dyes
Kinetics
Models, Molecular
Nucleic Acid Conformation
Protein Conformation
Replication Protein A
Signal Processing, Computer-Assisted
Spectrometry, Fluorescence
Xeroderma Pigmentosum Group D Protein
| | 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. | | Language | eng | | Pub Type(s) | Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
| | PubMed ID | 19748362 |
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