FIG. 2.

Putative TrwC targets in the human genome. (a) DNA sequences resembling the R388 nic site. The minimal region required for TrwC activity is highlighted in boldface. The nic site is indicated by a slash. Arrows show the inverted repeat recognized by TrwC. Nucleotides identical to the R388 sequence are shown in capital letters. The minimal target corresponds to the 173-190 oriT sequence present in plasmid pCIG1073. The mutation altering the R388 nic site in pCIG1110 is shown below the wild-type R388 sequence. Below, the seven human sequences, including the R388 14+2 sequence with a single mismach, are shown, together with shorter sequences mentioned in the text. Sequences are named indicating the human (Hu) chromosome number and the homologous nucleotides 5′+3′ with respect to the nic site; the mismatch position is indicated in parentheses. (b) TrwC binding assays on the (25+8) oligonucleotides containing the sequences shown in panel a. The amount of TrwC-N293 protein in each assay is indicated at the bottom (in nanograms). Arrows point to free (gray arrow) and retarded, TrwC-bound oligonucleotide (black arrow). (c) TrwC strand-transfer reactions on oligonucleotides, including the indicated R388 and human sequences. Arrows point to the cut product (gray arrow) or strand transfer product (black arrow), which is quantitated at the bottom of the gel (%ST). The assays for panels b and c were performed as described in Materials and Methods.