Fig. 1: DNA binding domain B (DBD-B) in RPA70 is phosphorylated by Aurora B kinase.

a Three RPA subunits RPA70, RPA32, and RPA14 form a heterotrimer and harbor multiple oligosaccharide/oligonucleotide (OB) domains. A, B, C, and D are DNA binding domains (DBDs). OB-F and the wh-domain are two protein interaction domains. The N-terminus of RPA32 (shown in red square) is hyperphosphorylated by multiple kinases including ATM, DNA-PK, CDK, and ATR and the known sites of phosphorylation are denoted in the insert. Arg-382 and Ser-384 in the putative Aurora B kinase motif are also noted in RPA70. The structure of human RPA OB-domains and connecting linkers were generated using AlphaFold and aligned to the trimerization core observed in the crystal structure (PDB:1L1O). b Sequence alignment of residues in DBD-B reveal a conserved putative phosphorylation motif for Aurora kinase in eukaryotes. A sequence logo representation of the conservation is also shown. c In vitro phosphorylation of recombinant RPA with Aurora B, followed by MS-MS analysis, shows residue Ser-384 in RPA70 as the sole site of phosphorylation. Extracted Ion Chromatograms (EICs) for unphosphorylated and phosphorylated tryptic peptide containing Ser-384 in the WT-RPA70 along with EIC of corresponding point mutations are shown. Alanine substitution of the Ser-384 phosphosite, or perturbation of the Aurora B recognition motif through a cancer-associated Gln substitution at Arg-382, results in loss of phosphorylation.