Properties and efficient scrap-and-build repairing of mechanically sheared 3’ DNA ends

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. Ohtsubo et al. show that mechanically sheared DNA 3’ terminus has 47% phosphate group, 20% hydroxyl group with remaining as abnormal termini, half of which is not repaired with T4 DNA polymerase treatment. They devise a method, scrap and build or SB-repairing, to efficiently repair 3’ DNA ends by combining phosphatase and exonuclease (scrap) followed by T4 DNA polymerase (build) reactions.