10779/crick.12652421.v1 Amy L Cherry Amy L Cherry Timothy J Nott Timothy J Nott Geoffrey Kelly Geoffrey Kelly Stuart L Rulten Stuart L Rulten Keith W Caldecott Keith W Caldecott Stephen J Smerdon Stephen J Smerdon Versatility in phospho-dependent molecular recognition of the XRCC1 and XRCC4 DNA-damage scaffolds by aprataxin-family FHA domains The Francis Crick Institute 2020 DNA break repair DNA-damage signalling FHA domain Amino Acid Sequence Binding Sites Casein Kinase II Crystallography, X-Ray DNA Damage DNA Repair DNA Repair Enzymes DNA-(Apurinic or Apyrimidinic Site) Lyase DNA-Binding Proteins Humans Molecular Sequence Data Nuclear Magnetic Resonance, Biomolecular Nuclear Proteins Phosphorylation Phosphotransferases (Alcohol Group Acceptor) Poly-ADP-Ribose Binding Proteins Protein Structure, Tertiary X-ray Repair Cross Complementing Protein 1 Smerdon U117584228 Developmental Biology 0601 Biochemistry and Cell Biology 2020-07-14 13:25:00 Journal contribution https://crick.figshare.com/articles/journal_contribution/Versatility_in_phospho-dependent_molecular_recognition_of_the_XRCC1_and_XRCC4_DNA-damage_scaffolds_by_aprataxin-family_FHA_domains/12652421 Aprataxin, aprataxin and PNKP-like factor (APLF) and polynucleotide kinase phosphatase (PNKP) are key DNA-repair proteins with diverse functions but which all contain a homologous forkhead-associated (FHA) domain. Their primary binding targets are casein kinase 2-phosphorylated forms of the XRCC1 and XRCC4 scaffold molecules which respectively coordinate single-stranded and double-stranded DNA break repair pathways. Here, we present the high-resolution X-ray structure of a complex of phosphorylated XRCC4 with APLF, the most divergent of the three FHA domain family members. This, combined with NMR and biochemical analysis of aprataxin and APLF binding to singly and multiply-phosphorylated forms of XRCC1 and XRCC4, and comparison with PNKP reveals a pattern of distinct but overlapping binding specificities that are differentially modulated by multi-site phosphorylation. Together, our data illuminate important differences between activities of the three phospho-binding domains, in spite of a close evolutionary relationship between them.