Sealing of gaps in duplex DNA by T4 DNA ligase
Open Access
- 11 March 1982
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 10 (5), 1425-1437
- https://doi.org/10.1093/nar/10.5.1425
Abstract
Single-strand gaps in DNA molecules were found to be a substrate for T4 DNA ligase. Sealing of the gaps was optimal at the same conditions as ligation of blunt-ended DNA molecules. Spermidine at a concentration of 2 mM stimulated the ligation of gaps, as well as the joining of DNA molecules with cohesive and blunt ends. In addition, spermidine reduced the optimal ATP concentration. The ligation of single-strand gaps was a slow process, reaching a plateau after several hours at 25°C. Approximately 10% of circular duplex plasmid pBR322 DNA molecules with a gap of 1–5 nucleotides could be converted to a covalently closed form. When such molecules were used for transformation of E. coli cells deletion mutants were obtained at a high frequency. The size and position of the gaps and the deletions were equivalent, confirming that T4 DNA ligase was sealing the gaps.Keywords
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