Double-strand break repair based on short-homology regions is suppressed under terminal deoxynucleotidyltransferase expression, as revealed by a novel vector system for analysing DNA repair by nonhomologous end joining

So Maezawa; Saori Nakano; Takaaki Kuniya; Osamu Koiwai; Kotaro Koiwai

doi:10.1002/2211-5463.12001

Double-strand break repair based on short-homology regions is suppressed under terminal deoxynucleotidyltransferase expression, as revealed by a novel vector system for analysing DNA repair by nonhomologous end joining

FEBS Open Bio. 2016 Jan 4;6(1):16-23. doi: 10.1002/2211-5463.12001. eCollection 2016 Jan.

Authors

So Maezawa¹, Saori Nakano¹, Takaaki Kuniya¹, Osamu Koiwai¹, Kotaro Koiwai¹

Affiliation

¹ Department of Applied Biological Science Faculty of Science and Technology Tokyo University of Science Noda Chiba Japan.

Abstract

We have constructed a novel, nonhomologous end-joining (NHEJ) assay vector (NAV), containing mKate2, Venus and ccdB genes. Cotransfection of NAV with a construct expressing the restriction enzyme I-SceI generated a double-strand break (DSB) in NAV that excised mKate2 and ccdB. Repair of this DSB produced an intact vector that expressed Venus, a green fluorescent protein. Because cells bearing the repaired NAV lacked the ccdB gene which slows cell proliferation, the cultures were enriched in cells containing repaired DSBs. DNA sequence analysis of the DSB junctions indicated that the repair was carried out mainly by using the closest homology sequence. Use of the NAV yielded rapid results within 3 days after transfection. We then used the NAV to analyse NHEJ in cells overexpressing terminal deoxynucleotidyltransferase (TdT). The results indicated that TdT suppresses DNA repair that is based on short (one- or two-base) homology regions, to efficiently add deoxynucleotides during VDJ recombination in lymphoid cells.

Keywords: DNA double‐strand break; DNA polymerase X; DNA polymerase λ; DNA polymerase μ; nonhomologous end joining; terminal deoxynucleotidyltransferase.