Supplementary material for Pommier et al., Proc. Natl. Acad. Sci. USA, 10.1073/pnas.190312697

Fig. 11. The trans-S oligonucleotide shown in Fig. 2A was either labeled at the 3'-end with [a-³²P]cordycepin (*A) (lanes 1-3) or 5'-end-phosphorylated (*) (lanes 4-6) with [g-³²P]ATP in the presence of T4 polynucleotide kinase [Pommier, Y., Kohlhagen, G., Pourquier, P., Sayer, J. M., Kroth, H. & Jerina, D. M. (2000) Proc. Natl. Acad. Sci. USA 97, 2040-2045]. Reactions were performed for 30 min at 25°C and were stopped by adding SDS (0.5% final concentration). Proteinase K was then added to the indicated samples and proteolytic digestion was performed for 1 h at 50°C. The size of the fragments (in bases) generated by top1 with the 3'-end-labeled oligonucleotide (including the cordycepin label) is indicated to the left of the gel. With the 5'-end-labeled DNA, the top1 cleavage complexes remain in the well of the gel in the absence of proteinase K (Top1-DNA). In the presence of proteinase K, the top1-linked DNA fragments are released into the gel with small residual top1 peptide attached because of incomplete proteinase K digestion (C = cleaved DNA with residual top1 peptide). The products labeled R correspond to recombinant DNA molecules resulting from top1-mediated illegitimate recombination [Pommier et al. (15)].