Abstract
Using synthetic oligodeoxynucleotides with 3'-OH ends and 32P-labelled 5'-phosphate ends and the technique of polyacrylamide gel electrophoresis, it is shown that, in the presence of the complementary polynucleotide, an AP (apurinic or apyrimidinic) site at the 3' or the 5' end of the labelled oligodeoxynucleotides does not prevent their ligation by T4 DNA ligase, although the reaction rate is decreased. This decrease is more severe when the AP site is at the 3' end; the activated intermediates accumulate showing that it is the efficiency of the adenyl-5'-phosphate attack by the 3'-OH of the base-free deoxyribose which is mostly perturbed. Using the same technique, it is shown that a mispaired base at the 3' or 5' end of oligodeoxynucleotides does not prevent their ligation. A one-nucleotide gap, limited by 3'-OH and 5'-phosphate, can also be closed by T4 DNA ligase although with difficulty; here again the activation of the 5'-phosphate end does not seem to be slowed down, but rather the 3'-OH attack of the adenyl-5'-phosphate. All these anomalous ligations take place with the nick or the gap in front of a continuous complementary strand. Blunt ends ligation of correct duplexes occurs readily; however an AP site or a mispaired base at the 3' or 5' end of one strand of the duplexes prevents ligation between these strands. But a missing nucleotide (responsible for one unpaired nucleotide protruding at the 3' or 5' end of the complementary strand) does not stop ligation of the shorter oligodeoxynucleotides between independent duplexes.
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- Bailly V., Verly W. G. Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst. Biochem J. 1987 Mar 1;242(2):565–572. doi: 10.1042/bj2420565. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goffin C., Verly W. G. T4 DNA ligase can seal a nick in double-stranded DNA limited by a 5'-phosphorylated base-free deoxyribose residue. Nucleic Acids Res. 1983 Nov 25;11(22):8103–8109. doi: 10.1093/nar/11.22.8103. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mosbaugh D. W., Linn S. Further characterization of human fibroblast apurinic/apyrimidinic DNA endonucleases. The definition of two mechanistic classes of enzyme. J Biol Chem. 1980 Dec 25;255(24):11743–11752. [PubMed] [Google Scholar]
- Oka J., Ueda K., Hayaishi O. Snake venom phosphodiesterase: simple purification with Blue Sepharose and its application to poly(ADP-ribose) study. Biochem Biophys Res Commun. 1978 Feb 28;80(4):841–848. doi: 10.1016/0006-291x(78)91321-9. [DOI] [PubMed] [Google Scholar]
- Streisinger G., Okada Y., Emrich J., Newton J., Tsugita A., Terzaghi E., Inouye M. Frameshift mutations and the genetic code. This paper is dedicated to Professor Theodosius Dobzhansky on the occasion of his 66th birthday. Cold Spring Harb Symp Quant Biol. 1966;31:77–84. doi: 10.1101/sqb.1966.031.01.014. [DOI] [PubMed] [Google Scholar]
- TAMM C., HODES M. E., CHARGAFF E. The formation apurinic acid from the desoxyribonucleic acid of calf thymus. J Biol Chem. 1952 Mar;195(1):49–63. [PubMed] [Google Scholar]
- Tsiapalis C. M., Narang S. A. On the fidelity of phage T4-induced polynucleotide ligase in the joining of chemically synthesized deoxyribooligonucleotides. Biochem Biophys Res Commun. 1970 May 22;39(4):631–636. doi: 10.1016/0006-291x(70)90251-2. [DOI] [PubMed] [Google Scholar]