d(GA x TC)(n) microsatellite DNA sequences enhance homologous DNA recombination in SV40 minichromosomes

Abstract

The genomic distribution of the abundant eukaryotic d(GA x TC)(n) DNA microsatellite suggests that it could contribute to DNA recombination. Here, it is shown that this type of microsatellite DNA sequence enhances DNA recombination in SV40 minichromosomes, the rate of homologous DNA recombination increasing by as much as two orders of magnitude in the presence of a d(GA x TC)(22) sequence. This effect depends on the region of the SV40 genome at which the d(GA x TC)(22) sequence is cloned. It is high when the sequence is located proximal to the SV40 control region but no effect is observed when located 3.5 kb away from the SV40 ori. These results indicate that the recombination potential of d(GA x TC)(n) sequences is likely linked to DNA replication and/or transcription. The potential contribution of the structural properties of d(GA x TC)(n) sequences to this effect is discussed.

Figure 1

Genomic organization of the constructs used in these experiments. All constructs contained a 2.6 kb long non-viral DNA insert flanked by 61 bp long direct repeats (HS), one of which is preceded by a repeated DNA sequence (RS). The BglI site at nucleotide position 0/5243 marks the position of the SV40 ori. See Materials and Methods for details.

Figure 2

(A) The plaquing efficiency, expressed as number of plaques per ng of transfected DNA, of pASV[GA] (circles), pASV[CA] (triangles) and pASV[0] (squares) is presented as a function of increasing days of post-transfection. (B) The plaquing efficiency at day 12 post-transfection is presented for pASV[GA], pASV[CA] and pASV[0].

Figure 3

(Top) SV40 recombinants arising from homologous DNA recombination across the HS sequences contained a single HS sequence flanked by two XbaI sites. Cleavage with XbaI and HindIII gave rise to RI and RII fragments of 500 and 700 bp in length, respectively (see text for details). (Bottom) The XbaI–HindIII restriction patterns are presented for five independent recombinants (lanes 1–5) arising from pASV[0], pASV[CA] and pASV[GA]. Lanes C correspond to the restriction patterns obtained from the original vectors and the arrows indicate the positions corresponding to fragments RI and RII.

Figure 4

(A) The plaquing efficiency of pASVt[GA] (circles), pASVt[CA] (triangles) and pASVt[0] (squares) is presented as a function of increasing days of post-transfection. (B) The plaquing efficiency of pASV3′[TC] (triangles), pASV[GA] (circles) and pASV[0] (squares) is presented as a function of increasing days of post-transfection.