Visualizing helicases unwinding DNA at the single molecule level.
DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes.
Fili
Natali
N
MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
Mashanov
Gregory I
GI
Toseland
Christopher P
CP
Batters
Christopher
C
Wallace
Mark I
MI
Yeeles
Joseph T P
JT
Dillingham
Mark S
MS
Webb
Martin R
MR
Molloy
Justin E
JE
D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000032:analysis* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis; D005090:Exodeoxyribonucleases / Q000032:analysis; D055767:Immobilized Proteins / Q000032:analysis; D008856:Microscopy, Fluorescence / Q000379:methods*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D055767:Immobilized Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D005090:Exodeoxyribonucleases; C407424:AddAB enzyme; D004265:DNA Helicases