T-DNA integration in Arabidopsis chromosomes. Presence and origin of filler DNA sequences

To investigate the relationship between T-DNA integration and double-stranded break (DSB) repair in Arabidopsis, we studied 67 T-DNA/plant DNA junctions and 13 T-DNA/T-DNA junctions derived from transgenic plants. Three different types of T-DNA-associated joining could be distinguished. A minority of T-DNA/plant DNA junctions were joined by a simple ligation-like mechanism, resulting in a junction without microhomology or filler DNA insertions. For about one-half of all analyzed junctions, joining of the two ends occurred without insertion of filler sequences. For these junctions, microhomology was strikingly combined with deletions of the T-DNA ends. For the remaining plant DNA/T-DNA junctions, up to 51-bp-long filler sequences were present between plant DNA and T-DNA contiguous sequences. These filler segments are built from several short sequence motifs, identical to sequence blocks that occur in the T-DNA ends and/or the plant DNA close to the integration site. Mutual microhomologies among the sequence motifs that constitute a filler segment were frequently observed. When T-DNA integration and DSB repair were compared, the most conspicuous difference was the frequency and the structural organization of the filler insertions. In Arabidopsis, no filler insertions were found at DSB repair junctions. In maize (Zea mays) and tobacco (Nicotiana tabacum), DSB repair-associated filler was normally composed of simple, uninterrupted sequence blocks. Thus, although DSB repair and T-DNA integration are probably closely related, both mechanisms have some exclusive and specific characteristics.