Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI.
Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investigated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA containing two endonuclease recognition sites spaced a few 100 bp apart connect small polystyrene beads to a glass surface. The position of a bead is tracked through video microscopy. Protein-mediated looping and unlooping is then observed as a sudden specific change in Brownian motion of the bead. With this method we are able to directly follow DNA looping kinetics of single protein-DNA complexes to obtain loop stability and loop formation times. We show that, in the absence of divalent cations, NaeI induces DNA loops of specific size. In contrast, under these conditions NarI mainly creates non-specific loops, resulting in effective DNA compaction for higher enzyme concentrations. Addition of Ca2+ increases the NaeI-DNA loop lifetime by two orders of magnitude and stimulates specific binding by NarI. Finally, for both enzymes we observe exponentially distributed loop formation times, indicating that looping is dominated by (re)binding the second recognition site.
van den Broek
Bram
B
Faculty of Sciences, Department of Physics and Astronomy and Laser Centre, De Boelelaan 1081, Vrije Universiteit Amsterdam, 1081 HV, The Netherlands.
Vanzi
Francesco
F
Normanno
Davide
D
Pavone
Francesco S
FS
Wuite
Gijs J L
GJ
D002118:Calcium / Q000494:pharmacology; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation
D004247:DNA; C060630:endodeoxyribonuclease NaeI; D015252:Deoxyribonucleases, Type II Site-Specific; C046719:GGCGCC-specific type II deoxyribonucleases; D002118:Calcium