Structural mechanism of TopBP1 recognizing phosphorylated HTATSF1 in homologous recombination repair.

Homologous recombination (HR) is a high-fidelity pathway for repairing DNA double-strand breaks, with the replication protein A (RPA)-Rad51 exchange representing a critical step. We previously reported that phosphorylated HIV-1 transactivator of transcription specific factor 1 (HTATSF1) interacts directly with DNA topoisomerase 2-binding protein 1 (TopBP1) and recruits it to DNA damage sites to promote RPA-Rad51 exchange and HR repair. Here, we demonstrate that TopBP1 cooperatively binds phosphorylated HTATSF1 through its BRCT1 and BRCT2 domains. We present the crystal structure of TopBP1 BRCT0-2 in complex with the phosphorylated extreme C terminus (ECT) peptide of HTATSF1, identifying key residues in BRCT1 involved in recognition. Notably, the N-terminal segment of the ECT peptide adopts a unique conformation, enabling specific hydrophobic interactions with V158 in BRCT1. Mutagenesis and binding assays confirm V158 as a key determinant allowing TopBP1 BRCT1 to discriminate HTATSF1 from other phosphorylated ligands. Furthermore, AlphaFold3 modeling combined with biochemical validation indicates that the ECT peptide also engages BRCT2 domain via its conserved phospho-binding pocket. Our work provides a structural basis for the specific TopBP1-HTATSF1 interaction and advances the mechanistic understanding for HR regulation.