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Nucleic Acids Research, 2001, Vol. 29, No. 16 e79
© 2001 Oxford University Press

Real-time observation of a single DNA digestion by {lambda} exonuclease under a fluorescence microscope field

Shun-ichi Matsuura, Jun Komatsu, Ken Hirano, Hachiro Yasuda, Kazunori Takashima, Shinji Katsura and Akira Mizuno*

Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan

A fluorescence microscopy technique has been developed to visualize the behavior of individual DNA and protein molecules. Real-time direct observation of a single DNA molecule can be used to investigate the dynamics of DNA–protein interactions, such as the DNA digestion reaction by {lambda} exonuclease. In conventional methods it is impossible to analyze the dynamics of an individual {lambda} exonuclease molecule on a DNA because they can only observe the average behavior of a number of exonuclease molecules. Observation of a single molecule, on the other hand, can reveal processivity and binding rate of an individual exonuclease molecule. To evaluate the dynamics of {lambda} exonuclease, a stained {lambda} DNA molecule with one biotinylated terminal was fixed on an avidin-coated coverslip and straightened using a d.c. electric field. Microscopic observation of digestion of a straightened DNA molecule by {lambda} exonuclease revealed that the DNA digestion rate was ~1000 bases/s and also demonstrated high processivity.

* To whom correspondence should be addressed. Tel: +81 532 44 6904: Fax: +81 532 44 6929; Email: mizuno{at}eco.tut.ac.jp


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