N.BspD6I DNA nickase strongly stimulates template-independent synthesis of non-palindromic repetitive DNA by Bst DNA polymerase.
Highly efficient DNA synthesis without template and primer DNAs occurs when N.BspD6I DNA nickase is added to a reaction mixture containing deoxynucleoside triphosphates and the large fragment of Bst DNA polymerase. Over a period of 2 h, virtually all the deoxynucleoside triphosphates (dNTPs) become incorporated into DNA. Inactivation of N.BspD6I nickase by heating inhibits DNA synthesis. Optimal N.BspD6I activity is required to achieve high yields of synthesized DNA. Electron microscopy data revealed that the majority of DNA molecules have a branched structure. Cloning and sequencing of the fragments synthesized demonstrated that the DNA product mainly consists of multiple hexanucleotide non-palindromic tandem repeats containing nickase recognition sites. A possible mechanism is discussed that addresses template-independent DNA synthesis stimulated by N.BspD6I nickase.
Zyrina
Nadezhda V
NV
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino 142290, Moscow Region, Russia.
Zheleznaya
Lyudmila A
LA
Dvoretsky
Eugene V
EV
Vasiliev
Victor D
VD
Chernov
Andrei
A
Matvienko
Nicholas I
NI
D001483:Base Sequence; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000502:physiology*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000502:physiology*; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D018895:Microsatellite Repeats / Q000502:physiology
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D004259:DNA-Directed DNA Polymerase; D003850:Deoxyribonuclease I; C514554:BspD6I protein, E coli; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases