title: Single molecular investigation of DNA looping and aggregation by restriction endonuclease BspMI.
abstract: DNA looping and aggregation induced by restriction endonuclease BspMI are studied by atomic force microscopy (AFM) and magnetic tweezers (MT). With Ca(2+) substituted for the normal enzyme cofactor Mg(2+) and enzyme concentration below the critical concentration of 6 units/mL, AFM images of DNA-BspMI complex show that the number of binding and looping events increases with enzyme concentration. At the critical concentration 6 of units/mL, all the BspMI binding sites are saturated. It is worth noting that nonspecific BspMI binding to DNA at saturation concentration represents more than 8% of the total BspMI-DNA complexes directly observed in AFM images. Furthermore, we used MT to prove that additional loops can form when enzyme concentration is higher than its saturation valueand the complex is incubated for a long time (>2 hrs). We ascribe this phenomenon to the aggregation of enzymes. The force spectroscopy of the BspMI-DNA complex shows that the pulling force required to open the loop of the complex at less than saturation concentration has a peak at about 3 pN, which is lower than the force required to open additional loops due to enzyme aggregation at higher than saturation concentration (>6 pN).
authors: School of Physics and Electronic Information, Wenzhou University, Wenzhou 325035, China.
authors: Yanwei
authors: Y
authors: Wang
authors: School of Physics and Electronic Information, Wenzhou University, Wenzhou 325035, China.
authors: Shiyong
authors: S
authors: Ran
authors: School of Physics and Electronic Information, Wenzhou University, Wenzhou 325035, China.
authors: Guangcan
authors: G
authors: Yang
mesh_terms: D001360:Avidin / Q000737:chemistry; D010582:Bacteriophage lambda / Q000235:genetics; D002122:Calcium Chloride / Q000737:chemistry; D053837:DNA Cleavage; D004279:DNA, Viral / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation; D066329:Protein Aggregates; D011485:Protein Binding
chemical_list: D004279:DNA, Viral; D066329:Protein Aggregates; D001360:Avidin; C052649:endodeoxyribonuclease BspMI; D015252:Deoxyribonucleases, Type II Site-Specific; D002122:Calcium Chloride