Abstract
DNA synthesized in vitro using deoxynucleoside phosphorothioates as substrates is quite similar to normal DNA in its biochemical properties (Vosberg, H.P., and Eckstein, F. (1977) Biochemistry 16, 3633-3640). In order to investigate the effect of phosphorothioate groups in DNA on the cleavage pattern of restriction endonucleases phosphorothioate double-stranded, circular, replicative form of fd DNA was synthesized in vitro with Escherichia coli DNA polymerase I using native single-stranded DNA as template and mixtures of three normal nucleotides and one nucleoside phosphorothioate analogue as substrates. The double-stranded products were hybrids with respect to their phosphorothioate content. Restriction analysis of normal and phosphorothioate DNA with the restriction endonucleases Hae III, Bam HI, Hpa II, HindII, Alu I, and Taq I showed that the enzymes were inhibited to different degrees depending on which of the nucleotides was replaced by the phosphorothioate. Most significant, inhibition was seen throughout with those DNAs which contained a phosphorothioate exactly at the cleavage site. Phosphorothioate substitutions at other positions, but still within the recognition sequences, were, except for Alu I, not or weakly inhibitory. Phosphorothioate nucleotides not present in the recognition sequences did not affect at all the fragment patterns. The results show that recognition sequences of restriction endonucleases can be selectively protected against cleavage by base-specific introduction of phosphorothioate groups into DNA.
Highlights
DNA synthesized in vitro using deoxynucleoside one native unmodified strand anda complementary strand in phosphorothioatesas substrates is quite similarto nor- which one of the normal nucleotides was replaced by the mal DNA in its biochemical properties
Because of the known slow enzymatic hydrolysis of phosphorothioate internucleotidic linkages by snake venom phosphodiesterase [2] and the nucleases associated with E . coli DNA polymerase I [2, 3] as well as T4DNA polymerase [4], we decided to investigate the restriction cleavage pattern of maland phosphorothioate DNA withtherestriction phosphorothioate fd DNAs which had been synthesized in the endonucleases Hue 111, Bam HI, Hpa 11, HindII, A h I, presence of one of the four nucleotide analogues
E. coli DNA polymerase I (EC 2.7.7.7) with an activityof groups in DNA on the activity of restriction enzymes arose from previous results demonstratingessentially normal incorporation of deoxynucleoside phosphorothioates into DNA [1]
Summary
Due to mismatching, we take these results as indication that the primary cause for cleavage inhibition is the presence of Restriction by Hue 111Endonuclease-This enzyme rec- two phosphorothioate groups adjacent to the C residues in ognizes the nucleotide sequence 5"GGCC-3' and produces the cleavage sequence, with one being at the cleavage site blunt ended polynucleotides by cutting between G andC. Substitution of Restriction by Barn HI Endonuclease-This enzyme recother nucleotides does not change the distribution of frag- ognizes the sequence 5"GGATCC-3' and cleaves between the ments as compared to cleavage of normal DNA (Fig. 1). Increasing the concentration up to 10 units/ inhibition of this enzyme by the presence of phosphorothioate sample was not sufficientto produce the normal limit products groups in fd DNA is strong. This sequence occurs in normal fd RF once with T as pyrimidine and A as purine, respectively [12] Inhibition of this enzyme is seen only after substitution of dATP by dATPaS (Fig. 5). Restriction by Tag I Endonuclease-Tag I is active at 65 "C and cleaves within the recognition sequence 5'-TCGA3' between T and C It produces 10 fragments with normal fd RF DNA [12]. DGTPaS and dTTPaS both do not affect the activity of this enzymeat all.Again, a phosphorothioate group at thecleavage site is significantlymore operative than substitutionsat other locations
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