The HSV-1 single-strand annealing protein ICP8 (UL29) is essential for viral DNA replication and recombination. Although its overall architecture has been described, the molecular basis of single-stranded DNA (ssDNA) recognition was unknown. We report crystal structures of C-terminally truncated ICP8 (ICP8 60) bound to poly(dT) 25 or poly(dA) 25 ssDNA at 3.0 to 3.1 resolution, along with higher-resolution apo structures of surface-entropy reduction variants. ssDNA binds within the neck region between the head and shoulder domains, contacting conserved OB-fold residues via base-specific hydrogen bonds, -stacking and phosphate backbone interactions. In the poly(dT) 25 complex, coordination of a Zn 2+ ion stabiliz... More
The HSV-1 single-strand annealing protein ICP8 (UL29) is essential for viral DNA replication and recombination. Although its overall architecture has been described, the molecular basis of single-stranded DNA (ssDNA) recognition was unknown. We report crystal structures of C-terminally truncated ICP8 (ICP8 60) bound to poly(dT) 25 or poly(dA) 25 ssDNA at 3.0 to 3.1 resolution, along with higher-resolution apo structures of surface-entropy reduction variants. ssDNA binds within the neck region between the head and shoulder domains, contacting conserved OB-fold residues via base-specific hydrogen bonds, -stacking and phosphate backbone interactions. In the poly(dT) 25 complex, coordination of a Zn 2+ ion stabilizes the zinc finger motif; whereas, in the poly(dA) 25 complex, Zn 2+ displacement promotes disulfide bond formation that effectively locks the protein into an altered conformation. Microscale thermophoresis and label-free differential scanning fluorimetry reveal a strong preference for pyrimidine-rich sequences, with nanomolar affinity for poly(dT) 25 and micromolar for poly(dA) 25 . Structural modeling identified Y543, R576, R772, R793, Y988, and F998 as key DNA-contact residues. Alanine substitutions caused severe replication defects, particularly for R772A, Y988A, and F998A. ssDNA binding induces 26 displacement and 35 degree rotation of the C-terminal domain and ordering of flexible loops, suggesting a mechanism for cooperative filament assembly. These structures define the molecular determinants of ICP8 ssDNA recognition, reveal thymidine bias and provide a framework for targeting ICP8-mediated functions in herpesvirus replication.
