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.

Abstract

2'-Deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP [caS]) 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 ofa 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 mI is limited and is followed by SI nuclease treatment, double-stranded DNA fragments asymmetrically shortened from just one side are produced. In this way 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 SI nuclease treatments, gives rise to truncated forms that, upon circularization by blunt-end ligation, transform E. coli and replicate in vivo.It is often useful to decrease the length ofa given DNA fragment in a progressive, controlled manner (1-7). Two different schemes are currently employed for progressively varying the length of double-stranded DNA fragments. One is the use of exonuclease III followed by single-strand-specific S1 nuclease (1,2,4,5,7,8). Another scheme uses Bal 31 nuclease (3, 6), which simultaneously digests both 5' and 3' ends of a doublestranded DNA fragment (9). The primary disadvantage of these approaches is that the nucleases act simultaneously at both ends of the fragment so that desirable sequences at one end are not preserved.The Sp diastereomer of the nucleoside a-thiotriphosphates (which have a sulfur substituted for an oxygen at the a-phosphate) act as substrates for Escherichia coli DNA polymerase I in DNA synthesis (10). These compounds, although incorporated at an efficiency similar to that of the natural substrates, effectively block the 3' exonuclease activity of DNA polymerase I at the phosphorothioate nucleotide linkage (unpublished results). In work reported here we found that placement of an athionucleotide at only one of the 3' ends of a DNA fragment effectively blocks that end from digestion with exonuclease III. Digestion proceeds progressively from the opposite end and, after S1 nuclease treatment, the fragment is shortened with the integrity of the nucleotide sequence at the "capped" end preserved. Alternatively, if exonuclease III digestion is allowed to proceed to completion, a full-length single strand is generated from the original double strand.This work also demonstrates that, when 2'-deoxyadenosine 5'-O-(l-thiotriphosphate) (dATP [aS]) is inserted into 3' recessed ends ofrestriction fragments, the phosphorothioate linkage allows subsequent ligation ofthe DNA, restriction ofligated junctions, and in vivo replication of plasmids containing phosphorothioate nucleotide linkages. Purific...

Identification of a Universal B Cell Epitope on Dna Topoisomerase I, an Autoantigen Associated With Scleroderma

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Autoregulation of the human N-myc oncogene is disrupted in amplified but not single-copy neuroblastoma cell lines

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