Interaction between Conjugative and Retrotransposable Elements in Horizontal Gene Transfer
Figure 5
LtrB nicks ssDNA of its cognate oriT in sequence- and strand-specific manner.
(A) LtrB relaxase can cleave ssDNA oriT in strand-specific manner. 32P-labeled oligonucleotides, containing either pRS01 oriT (blue) or R388 oriT (grey), were incubated with LtrB-HIS6 (Lane 1), LtrB (Y21A)-HIS6 (Lane 2) or buffer control (Lane 3) in cleavage buffer and products were separated by denaturing PAGE gel alongside φ×174 DNA/Hinf1 marker (Lane M). A line marks the substrate band (96 nt). An arrow shows the cleavage band (61 nt). Cleavage was only observed with wild-type LtrB-HIS6 on pRS01 oriT, its native target (Lane 1, left panel). The orientation of arrows, representing oligonucleotides, reflects the strand of oriT, either sense or antisense. The asterisk is the site of labeling. (B) Mapping of nick site. After incubation with relaxase, primer extension reaction was performed using primer IDT3492, which is specific for the pGEM vector. The cleavage was observed within pRS01 oriT and the sequence of nic was established (vertical arrow on schematic, horizontal arrows beside autoradiogram). The ssDNA was prepared from ds substrates pONoriT1 and pONoriT2, and incubated with LtrB-HIS6 (Lane 1), LtrB (Y21A)-HIS6 (Lane 2) or buffer control (Lane 3). These reactions were used in primer extension assays and separated by denaturing PAGE, alongside sequencing ladders. The cleavage band at the nick site is only present with wild-type LtrB-HIS6 (Lane 1) on its native target, pRS01 oriT. No cleavage was observed within R388 oriT indicating specificity of LtrB relaxase for its cognate oriT.