Journal
COMMUNICATIONS BIOLOGY
Volume 2, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s42003-019-0660-7
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Funding
- Ministry of Education, Culture, Sports, Science, and Technology, Japan [15H04471]
- Institute for Fermentation, Osaka (IFO), Japan
- Grants-in-Aid for Scientific Research [15H04471] Funding Source: KAKEN
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Repairing of DNA termini is a crucial step in a variety of DNA handling techniques. In this study, we investigated mechanically-sheared DNA 3'-ends (MSD3Es) to establish an efficient repair method. As opposed to the canonical view of DNA terminus generated by sonication, we showed that approximately 47% and 20% of MSD3Es carried a phosphate group and a hydroxyl group, respectively. The others had unidentified abnormal terminal structures. Notably, a fraction of the abnormal 3' termini (about 20% of the total) was not repaired after the removal of 3' phosphates and T4 DNA polymerase (T4DP) treatment. To overcome this limitation, we devised a reaction, in which the 3'- > 5' exonuclease activity of exonuclease III (3'- > 5' exonuclease, insensitive to the 3' phosphate group) was counterbalanced by the 5'- >3' polymerase activity of T4DP. This combined reaction, termed SB-repairing (for scrap-and-build repairing), will serve as a useful tool for the efficient repair of MSD3Es.
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