We have carried out solution equilibrium binding studies of ICP8, the major single-stranded DNA (ssDNA)-binding protein of herpes simplex virus type I, in order to determine the thermodynamic parameters for its interaction with ssDNA. Fluorescence anisotropy measurements of a 5-fluorescein-labeled 32-mer oligonucleotide revealed that ICP8 formed a nucleoprotein filament on ssDNA with a binding site size of 10 nucleotides/ICP8 monomer, an association constant at 25°C, K ‫؍‬ 0.55 ؎ 0.05 ؋ 10 6 M ؊1, and a cooperativity parameter, ‫؍‬ 15 ؎ 3. The equilibrium constant was largely independent of salt, ␦log(K)/␦log([NaCl]) ‫؍‬ ؊2.4 ؎ 0.4. Comparison of these parameters with other ssDNA-binding proteins showed that ICP8 reacted with an unusual mechanism characterized by low cooperativity and weak binding. In addition, the reaction product was more stable at high salt concentrations, and fluorescence enhancement of etheno-ssDNA by ICP8 was higher than for other ssDNA-binding proteins. These last two characteristics are also found for protein-DNA complexes formed by recombinases in their active conformation. Given the proposed role of ICP8 in promoting strand transfer reactions, they suggest that ICP8 and recombinase proteins may catalyze homologous recombination by a similar mechanism.ICP8 is 128-kDa zinc metalloprotein coded by the UL29 gene of herpes simplex virus type 1 (HSV-1).1 It is one of seven virus-encoded proteins necessary for HSV-1 origin-dependent DNA replication (1). ICP8 forms an extended nucleoprotein filament with ssDNA in vitro; it binds more readily to ssDNA than to double-stranded DNA (1-6), and DNA binding may involve contacts by free sulfhydryl groups and surface lysine and tyrosine residues (6, 7). In addition to its role as an ssDNAbinding protein, complexes of ICP8 with other proteins may act during viral replication. ICP8 physically interacts via the UL8 protein with the HSV-1 helicase primase heterotrimer thereby stimulating unwinding of intact and damaged duplex DNA (8, 9); likewise a physical interaction between ICP8 and the herpes UL9 helicase has also been shown (10). Specific protein-protein interactions between ICP8, UL9 protein, and subunits of the DNA helicase primase are required for the assembly of these proteins into prereplicative sites, and recruitment of the DNA polymerase into this complex is mediated by the UL42 subunit (11,12).Cells infected with HSV-1 display a high frequency of homologous recombination (13). In vitro experiments suggest that ICP8 may be responsible for homologous recombination in vivo. ICP8 exhibits helix destabilizing and DNA renaturing activities (2, 14). The protein also catalyzes homologous pairing and strand exchange activity in vitro (15) where it is able to transfer a DNA strand from a linear duplex to a complementary single-stranded circular DNA. The reaction requires MgCl 2 but not (d)NTPs, and strand exchange is limited to a few 100 base pairs.Understanding the role of ICP8 in the DNA replication and recombination reactions of HSV-1 will require kn...