Structural basis of DNA recognition of tomato yellow leaf curl virus replication-associated protein

Conserved and multifunctional Geminivirus Replication-associated Protein (Rep) specifically recognizes the replication origin and initiates viral DNA replication. We report the X-ray crystallography-based structures of two complexes containing the N-terminal domain (5-117aa) of Tomato yellow leaf curl virus (TYLCV) Rep: the catalytically-dead Rep in complex with nonanucleotide ssDNA (Rep5-117 Y101F-ssDNA) as well as the catalytically-active phosphotyrosine covalent adduct (Rep(5-117)-ssDNA). These structures provide functional insight into the role of Rep in viral replication. Metal ions stabilize the DNA conformation by interacting with the phosphate group of adenine and thus promote formation of the catalytic center. Furthermore, we identified a compound that inhibits the binding of Rep to ssDNA and dsDNA and found that the addition of metal ions compromises the inhibitory effectiveness of this compound. This study demonstrates the mechanism of DNA recognition and cleavage process of viral Rep, emphasizing the role of metal ions.