Single-molecule analysis reveals differential effect of ssDNA-binding proteins on DNA translocation by XPD helicase.
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.
Honda
Masayoshi
M
Department of Biochemistry, University of Illinois at Urbana-Champaign, 61801, USA.
Park
Jeehae
J
Pugh
Robert A
RA
Ha
Taekjip
T
Spies
Maria
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002232:Carbocyanines; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D005456:Fluorescent Dyes; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D051716:Replication Protein A / Q000737:chemistry / Q000378:metabolism*; D012815:Signal Processing, Computer-Assisted; D013050:Spectrometry, Fluorescence; D051759:Xeroderma Pigmentosum Group D Protein / Q000737:chemistry / Q000378:metabolism*
D019843:Archaeal Proteins; D002232:Carbocyanines; D005456:Fluorescent Dyes; D051716:Replication Protein A; C089187:cyanine dye 3; C085321:cyanine dye 5; D000255:Adenosine Triphosphate; D004247:DNA; D051759:Xeroderma Pigmentosum Group D Protein