TY - JOUR
T1 - Properties and efficient scrap-and-build repairing of mechanically sheared 3’ DNA ends
AU - Ohtsubo, Yoshiyuki
AU - Sakai, Keiichiro
AU - Nagata, Yuji
AU - Tsuda, Masataka
N1 - Publisher Copyright:
© 2019, The Author(s).
PY - 2019/12/1
Y1 - 2019/12/1
N2 - 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.
AB - 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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U2 - 10.1038/s42003-019-0660-7
DO - 10.1038/s42003-019-0660-7
M3 - Article
C2 - 31925254
AN - SCOPUS:85074878541
SN - 2399-3642
VL - 2
JO - Communications Biology
JF - Communications Biology
IS - 1
M1 - 409
ER -