Figure 3

HinLVA mediates inversions of short and long DNA fragments. HinLVA-mediated flipping was assessed by NheI restriction digests. Gel electrophoresis images are shown in A and B. (A) A construct carrying hixC-flanked RBS-tetA(C) in the forward orientation (solid purple arrow) is characterized by a ~200 bp NheI restriction fragment (gel lane 2). The reverse orientation of RBS-tetA(C) (hatched purple arrow) yields a larger ~1100 bp band (lane 3). When forward oriented hixC-flanked RBS-tetA(C) is exposed to HinLVA, bands for both forward and reverse orientations of RBS-tetA(C) are detected (lane 4). (B) Similarly, forward oriented hixC-flanked pBAD promoter (solid green arrow) is converted to the reverse orientation (hatched green arrow) after exposure to HinLVA (lane 4). These data show that HinLVA-mediated inversion activity is not constrained by the length of hixC-flanked DNA.