Fig. S1.

Supercoiling retention assay with negatively supercoiled (A) or relaxed (B) DNA substrates. (A) Drosophila TDRD3 was added first to the reaction for DNA binding before the addition of Drosophila Top1 to relax topologically unconstrained supercoiling. Final reaction products after protein removal have a deficiency in linking number compared with the negatively supercoiled substrate (Upper row). Addition of ethidium bromide to the reaction generates products with more linking deficiency (Lower row). (B) Top1 was added first to relax the negatively supercoiled substrate, followed by the addition of TDRD3 to bind to single-stranded regions to produce slightly positively supercoiled intermediates and further Top1 relaxation to yield the final product. Following protein removal, the final reaction product will have a slight linking deficiency compared with the fully relaxed DNA.