Substrate properties of 25-nt parallel-stranded linear DNA duplexes.

Abstract

Four 25-nt oligonucleotides consisting of sequences of dA and dT (D1-4) have been synthesized. As shown in a companion paper (Rippe et al., 1989), the two combinations D1.D3 and D2.D4 form normal antiparallel duplexes, whereas the pairs D1.D2 and D3.D4 constitute duplexes with the same sequences, but with the two strands parallel to each other. The activities of the following DNA processing enzymes and chemical reagents on the parallel stranded (ps) and antiparallel stranded (aps) duplexes were tested. (i) The restriction endonucleases DraI, SspI, and MseI do not cut the ps duplexes. (ii) DNase I and exonuclease III exhibit a much lower activity with the ps duplexes. (iii) The nuclease activities of S 1 nuclease, micrococcal nuclease (S 7), phage lambda 5'-exonuclease, and the 3'-5' nuclease activity of Escherichia coli DNA polymerase I and its large fragment are higher with the ps than with the aps substrates. (iv) Bal 31 nuclease and the chemical nuclease 1,10-phenanthroline-copper ion [(OP)2Cu+] degrade ps-DNA and aps-DNA at approximately the same rate but show preferred cutting sites only with the aps molecules. (v) The iron(II)-EDTA complex has equivalent nuclease activities with the ps and the aps molecules. (vi) The ps duplex is not a substrate for blunt-end ligation with phage T4 DNA ligase.