Real-time observation of a single DNA digestion by lambda exonuclease under a fluorescence microscope field.
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 approximately 1000 bases/s and also demonstrated high processivity.
Matsuura
S
S
Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan.
Komatsu
J
J
Hirano
K
K
Yasuda
H
H
Takashima
K
K
Katsura
S
S
Mizuno
A
A
D001360:Avidin / Q000378:metabolism; D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics*; D001583:Benzoxazoles / Q000378:metabolism; D019921:Biotinylation; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004574:Electromagnetic Fields; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D007364:Intercalating Agents / Q000378:metabolism; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D011485:Protein Binding; D011806:Quinolinium Compounds / Q000378:metabolism; D013997:Time Factors; D014764:Viral Proteins
D001583:Benzoxazoles; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D011806:Quinolinium Compounds; D014764:Viral Proteins; D001360:Avidin; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D005090:Exodeoxyribonucleases; C043586:exo protein, Bacteriophage lambda