Real-time direct observation of single-molecule DNA hydrolysis by exonuclease III.
Single-molecule DNA digestion by exonuclease III, which has 3' to 5' exonuclease activity, was analyzed using a micro-channel with two-layer laminar flow. First, a DNA-bead complex was optically trapped in one layer in the absence of exonuclease III permitted the DNA to be stretched by the laminar flow. The exonuclease III reaction was initiated by moving the trapped DNA-bead complex to another layer of flow, which contained exonuclease III. As the reaction proceeded, the fluorescently-stained DNA was observed to shorten. The process was photographed; examination of the photographs showed that the DNA molecule shortened in a linear fashion with respect to the reaction time. The digestion rate obtained from the single-molecule experiment was compared to that measured from a bulk experiment and was found to be ca. 28 times higher than the bulk digestion rate.
Kurita
Hirofumi
H
Department of Ecological Engineering, Toyohashi University of Technology, Aichi 441-8580, Japan.
Inaishi
Ken-ichi
K
Torii
Ken
K
Urisu
Madoka
M
Nakano
Michihiko
M
Katsura
Shinji
S
Mizuno
Akira
A
D010582:Bacteriophage lambda / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006868:Hydrolysis; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D052898:Optical Tweezers
D004279:DNA, Viral; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III