title: 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.
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.
authors.affiliation: Department of Applied Biological Science Faculty of Science and Technology Tokyo University of Science Noda Chiba Japan.
authors.forename: So
authors.initials: S
authors.lastname: Maezawa
authors.affiliation: Department of Applied Biological Science Faculty of Science and Technology Tokyo University of Science Noda Chiba Japan.
authors.forename: Saori
authors.initials: S
authors.lastname: Nakano
authors.affiliation: Department of Applied Biological Science Faculty of Science and Technology Tokyo University of Science Noda Chiba Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Kuniya
authors.affiliation: Department of Applied Biological Science Faculty of Science and Technology Tokyo University of Science Noda Chiba Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Koiwai
authors.affiliation: Department of Applied Biological Science Faculty of Science and Technology Tokyo University of Science Noda Chiba Japan.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Koiwai
keywords: DNA double‐strand break; DNA polymerase X; DNA polymerase λ; DNA polymerase μ; nonhomologous end joining; terminal deoxynucleotidyltransferase