T-DNA integration into the Arabidopsis genome depends on sequences of pre-insertion sites

Abstract

A statistical analysis of 9000 flanking sequence tags characterizing transferred DNA (T-DNA) transformants in Arabidopsis sheds new light on T-DNA insertion by illegitimate recombination. T-DNA integration is favoured in plant DNA regions with an A-T-rich content. The formation of a short DNA duplex between the host DNA and the left end of the T-DNA sets the frame for the recombination. The sequence immediately downstream of the plant A-T-rich region is the master element for setting up the DNA duplex, and deletions into the left end of the integrated T-DNA depend on the location of a complementary sequence on the T-DNA. Recombination at the right end of the T-DNA with the host DNA involves another DNA duplex, 2-3 base pairs long, that preferentially includes a G close to the right end of the T-DNA.

Figure 1

Gene and FST densities along chromosome 3 of A. thaliana. Each peak is a number of FSTs (above) or genes (below) per 100 kb. The centromeric region is represented by a circle on the abscissa.

Figure 2

Deletions in the integrated T-DNA and A. thaliana sequences at IS. (A) Histogram of the number of FSTs classified on the basis of the ends of the sequence of the integrated T-DNA. 8525 and 394 FSTs were analysed for left and right borders, respectively. The canonical T-DNA sequence runs from (a) to (e). (B) The over-representation of nucleotides in the plant sequence before and after IS depends on the end of the integrated T-DNA. (a) to (f) indicate nicking sites as illustrated below the T-DNA sequence in (A). Over-represented nucleotides (shaded boxes in Table 1) are shown at five positions downstream and seven positions upstream of IS. (C) A deletion of 1 bp upstream of the IS can explain the over-representation of two different nucleotides at different positions in the case of a canonical integration (a). The sequence N(T)₅CAGGAN is taken as an example of a plant sequence around an IS. (D) Identity scores between the complement of the canonical T-DNA left border, CAGGA and sequences downstream of IS (triangles) or randomly taken from the A. thaliana genome (circles).

Figure 3

Relative base composition of the A. thaliana DNA at T-DNA IS. IS were mapped in the A. thaliana genome by sequence comparison between 4330 FSTs obtained from the T-DNA LB and not flanked by filler DNA.

Figure 4

A model of the T-DNA integration process. The given example is for a putative T-DNA that, as a consequence of its integration is deleted by six nucleotides in the 3′ LB [position (b) in Figure 2A] but complete in its RB. For convenience, only the upper strand of the host DNA is represented. A T-rich region [T(n)] operates as a preferential site of entry of the T-DNA LB. Starting from its 3′ end, the T-DNA scans the plant DNA until it finds a microcomplementarity just downstream of the T-rich region. A nick (2) is generated in the host DNA downstream of the microcomplementarity-based duplex and used as a priming site to synthesize the complementary strand of the T-DNA until the 5′ RB covalently linked to VirD2 is reached. The integration process of the newly synthesized doublestranded DNA frequently brings about a deletion in the host DNA. Recombination between the host bottom strand and the T-DNA operates at sites 1 and 3 after the action of exonucleases on both ends of the T-DNA.