The accessibility of thiophosphorylated groups in DNA fragments to the enzymatic activity of ligases and restriction endonuclease Bbs I.
The aim of this paper was to test the possibility to ligate and hydrolyse DNA sequences containing thiomodified ends and bonds. T4 DNA ligase was shown to ligate DNA fragments regardless of whether it contains phosphorylated or thiophosphorylated 5'-end. But the cleavage of an internally thiomodified phosphodiester bond was found to be totally inhibited when using the non-palindromic restrictase Bbs I. The special properties of this restriction endonuclease should allow the development of an oriented cloning strategy when combined with T4 ligase and a thiophosphorylation of DNA fragments.
Schenk
J A
JA
Institute for Medical Immunology, Charite, Humboldt University, Berlin-Buch, Germany.
Heymann
S
S
Micheel
B
B
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism*; D011088:DNA Ligases / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D013873:Thionucleotides / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D013873:Thionucleotides; D004247:DNA; D004262:DNA Restriction Enzymes; D011088:DNA Ligases