T-DNA Integration in Arabidopsis Chromosomes. Presence and Origin of Filler DNA Sequences  

Abstract

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.

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

“…This filler DNA showed no similarity to any other known DNA. Scrambled filler DNA is often observed at the integration sites of plasmid and T-DNA molecules in transgenic plants (Gorbunova and Levy, 1997;Kumar and Fladung, 2000;Makarevitch et al, 2003;Windels et al, 2003). In their report, Gorbunova and Levy (1997) proposed that the origin of scrambled DNA may involve the invasion of ectopic templates and multiple template switches during DNA integration.…”

The T‐DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

“…New DNA is synthesized before and during integration of the T-DNA because the single-stranded T-strand becomes double stranded shortly after transfer (Chilton and Que, 2003;Tzfira et al, 2003). Additionally, filler sequences between the T-DNA and the target DNA result from new DNA synthesis, because these filler DNAs consist of mosaic sequences derived from the T-DNA ends and plant target sequences (Windels et al, 2003;Mü ller et al, 2007). These mechanisms involved in the new DNA synthesis during integration might possibly also trigger replication of the complete T-DNA via strand switching and/or rolling circlelike T-DNA replication.…”

“…Integration of T-DNA is often accompanied by small deletions of the plant DNA target site and/or the ends of the invading T-DNA. Microhomology is commonly found between the plant pre-insertion site and T-DNA ends, and filler DNA is frequently present at the T-DNA/plant junctions (Windels et al, 2003;Tzfira et al, 2004;Mü ller et al, 2007;Ziemienowicz et al, 2008).…”

Simple and complex nuclear loci created by newly transferred chloroplast DNA in tobacco

“…A further 13-bp nuclear sequence upstream (dotted line above the residues in Fig. 3) also was similar to the 3Ј terminus of the ptDNA sequence of kr18-2 except for two nucleotide substitutions, suggesting that the synthesis of short segments from neighboring ptDNA or nuclear DNA may have been involved in the integration process (27,28). In addition, an inverted 188-bp sequence derived from neoSTLS2 with some rearrangements (a 6-bp deletion of the neoSTLS2 sequence and a 10-bp insertion of an unknown sequence) was located 463 bp downstream of J17-2a (Fig.…”