A two-hybrid system was used to isolate factors that interact with the C-terminal region of DNA topoisomerase IIbeta. A positive clone isolated from a HeLa cDNA library encoded 1522 amino acid residues (molecular mass 170670). The protein, designated topoisomerase-IIbeta-binding protein 1 (TopBP1), interacted with the C-terminal region of topoisomerase IIbeta synthesized in vitro. A database search indicated that TopBP1 possessed eight regions similar to regions of Rad4, Cut5, Ect2, Rev1 and X-ray repair cross-complementing 1 (XRCC1) proteins and a region similar to auto-modification sites of poly(ADP-ribose) polymerase, suggesting that TopBP1 supported catalytic reactions of topoisomerase II through transient breakages of DNA strands.