Structural basis for DNA 3′-end processing by human tyrosyl-DNA phosphodiesterase 1

Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA 3′-end processing enzyme that repairs topoisomerase 1B-induced DNA damage. We use a new tool combining site-specific DNA–protein cross-linking with mass spectrometry to identify Tdp1 interactions with DNA. A conserved phenylalanine (F259) of Tdp1, required for efficient DNA processing in biochemical assays, cross-links to defined positions in DNA substrates. Crystal structures of Tdp1–DNA complexes capture the DNA repair machinery after 3′-end cleavage; these reveal how Tdp1 coordinates the 3′-phosphorylated product of nucleosidase activity and accommodates duplex DNA. A hydrophobic wedge splits the DNA ends, directing the scissile strand through a channel towards the active site. The F259 side-chain stacks against the −3 base pair, delimiting the junction of duplexed and melted DNA, and fixes the scissile strand in the channel. Our results explain why Tdp1 cleavage is non-processive and provide a molecular basis for DNA 3′-end processing by Tdp1.