Probing Interactions within the synaptic DNA-SfiI complex by AFM force spectroscopy.
SfiI belongs to a family of restriction enzymes that function as tetramers, binding two recognition regions for the DNA cleavage reaction. The SfiI protein is an attractive and convenient model for studying synaptic complexes between DNA and proteins capable of site-specific binding. The enzymatic action of SfiI has been very well characterized. However, the properties of the complex before the cleavage reaction are not clear. We used single-molecule force spectroscopy to analyze the strength of interactions within the SfiI-DNA complex. In these experiments, the stability of the synaptic complex formed by the enzyme and two DNA duplexes was probed in a series of approach-retraction cycles. In order to do this, one duplex was tethered to the surface and the other was tethered to the probe. The complex was formed by the protein present in the solution. An alternative setup, in which the protein was anchored to the surface, allowed us to probe the stability of the complex formed with only one duplex in the approach-retraction experiments, with the duplex immobilized at the probe tip. Both types of complexes are characterized by similar rupture forces. The stability of the complex was determined by measuring the dependence of rupture forces on force loading rates (dynamic force spectroscopy) and the results suggest that the dissociation reaction of the SfiI-DNA complex has a single energy barrier along the dissociation path. Dynamic force spectroscopy was instrumental in revealing the role of the 5 bp spacer region within the palindromic recognition site on DNA-SfiI in the stability of the complex. The data show that, although the change of non-specific sequence does not alter the position of the activation barrier, it changes values of the off rates significantly.
Krasnoslobodtsev
Alexey V
AV
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Shlyakhtenko
Luda S
LS
Lyubchenko
Yuri L
YL
D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism* / Q000648:ultrastructure; D004800:Enzymes, Immobilized / Q000378:metabolism; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods*; D011485:Protein Binding
D004268:DNA-Binding Proteins; D004800:Enzymes, Immobilized; D004247:DNA; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific