Single-Molecule Studies of Restriction Endonuclease Kinetics
نویسندگان
چکیده
منابع مشابه
Single molecule detection of DNA looping by NgoMIV restriction endonuclease.
Single molecule fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy were used to investigate DNA looping by NgoMIV restriction endonuclease. Using a linear double-stranded DNA (dsDNA) molecule labeled with a fluorescence donor molecule, Cy3, and fluorescence acceptor molecule, Cy5, and by varying the concentration of NgoMIV endonuclease from 0 to 3 x 10(-6) M...
متن کاملExploring both sequence detection and restriction endonuclease cleavage kinetics by recognition site via single-molecule microfluidic trapping.
We demonstrate the feasibility of a single-molecule microfluidic approach to both sequence detection and obtaining kinetic information for restriction endonucleases on dsDNA. In this method, a microfluidic stagnation point flow is designed to trap, hold, and linearize double-stranded (ds) genomic DNA to which a restriction endonuclease has been pre-bound sequence-specifically. By introducing th...
متن کاملCharacterization of steady state, single-turnover, and binding kinetics of the TaqI restriction endonuclease.
The TaqI restriction endonuclease recognizes and cleaves the duplex DNA sequence T decreases CGA. Steady state kinetic analysis with a small oligodeoxyribonucleotide substrate showed that the enzyme obeyed Michaelis-Menten kinetics (Km = 53 nM, kcat = 1.3 min-1 at 50 degrees C and Km = 0.5 nM, kcat = 2.9 min-1 at 60 degrees C). At 0 degree C, the enzyme was completely inactive, while at 15 degr...
متن کاملCovalent joining of the subunits of a homodimeric type II restriction endonuclease: single-chain PvuII endonuclease.
The PvuII restriction endonuclease has been converted from its natural homodimeric form into a single polypeptide chain by tandemly linking the two subunits through a short peptide linker. The arrangement of the single-chain PvuII (sc PvuII) is (2-157)-GlySerGlyGly-(2-157), where (2-157) represents the amino acid residues of the enzyme subunit and GlySerGlyGly is the peptide linker. By introduc...
متن کاملSingle-Molecule Kinetics of Interfacial Electron Transfer
Measurements of single-molecule chemical reaction kinetics are demonstrated for interfacial electron transfer from excited cresyl violet molecules to the conduction band of indium tin oxide (ITO) or energetically accessible surface electronic states under ambient conditions by using a far-field fluorescence microscope. In this system, each single molecule exhibits a single-exponential electron ...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
ژورنال
عنوان ژورنال: Biophysical Journal
سال: 2012
ISSN: 0006-3495
DOI: 10.1016/j.bpj.2011.11.2663