A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo.
2'-Deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP[alpha S]) was introduced into the 3' ends of DNA restriction fragments with Escherichia coli DNA polymerase I to give phosphorothioate internucleotide linkages. Such "capped" 3' ends were found to be resistant to exonuclease III digestion. Moreover, the resistance to digestion is great enough that, under conditions used by us, just one strand of a double helix is digested by exonuclease III when a cap is placed at only one end; when digestion is carried to completion, this results in production of intact single strands. When digestion with exonuclease III is limited and is followed by S1 nuclease treatment, double-stranded DNA fragments asymmetrically shortened from just one side are produced. In this was thousands of nucleotides can be selectively removed from one end of a restriction fragment. In vitro introduction of phosphorothioate linkages into one end of a linearized replicative plasmid, followed by exonuclease III and S1 nuclease treatments, gives rise to truncated forms that, upon circularization by blunt-end ligation, transform E. coli and replicate in vivo.
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004261:DNA Replication; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism*; D005090:Exodeoxyribonucleases; D005092:Exonucleases / Q000378:metabolism*; D010957:Plasmids; D013379:Substrate Specificity; D013873:Thionucleotides / Q000378:metabolism*
D004277:DNA, Single-Stranded; D003838:Deoxyadenine Nucleotides; D013873:Thionucleotides; C020613:2'-deoxyadenosine 5'-O-(2-thiotriphosphate); D003851:Deoxyribonucleases; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C020133:exodeoxyribonuclease III; D004262:DNA Restriction Enzymes