Equilibrium Binding of Single-stranded DNA with Herpes Simplex Virus Type I-coded Single-stranded DNA-binding Protein, ICP8

Abstract

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...

The Herpes Simplex Virus Type-1 Single-strand DNA-binding Protein (ICP8) Promotes Strand Invasion

“…E. coli RecA and T4 UvsX protein) than they are to other SSBs (e.g. E-SSB, RP-A, and T4 gp32) (9). Thus, like bona fide recombinases, ICP8 stretches ssDNA, possibly to facilitate the search for homology, forms complexes with ssDNA that are stable at high salt concentrations and exhibits both weaker and lower cooperativity ssDNA binding than other SSBs.…”

“…1A, ICP8 results in the assimilation of the 100-mer into homologous form I acceptor DNA, presumably leading to the formation of a D-loop (lane 1). The concentration of ICP8 used was 2.5-fold in excess to that required to coat the 100-mer (9). Under these conditions, up to 15% of the form I acceptor DNA participated in D-loop formation.…”

In Vitro Strand Exchange Promoted by the Herpes Simplex Virus Type-1 Single Strand DNA-binding Protein (ICP8) and DNA Helicase-Primase

“…This may be rationalized by the fact that the substrates used in our current work provided ICP8 with an appropriate loading site (25).…”

“…The substrates for this reaction were acceptor DNA, consisting of circular M13 ssDNA, and donor DNA, consisting of 5Ј 32 P-labeled complementary linear dsDNA molecules. DNA substrates possessing TspRI ends (9 nucleotide 3Ј overhang) or cos termini (12 nucleotide 5Ј overhang) were used to supply ICP8 with an appropriate loading site (25). In each case, the strand possessing the overhang was complementary to circular M13 ssDNA.…”

“…ICP8 binds preferentially to single-stranded (ss) DNA with a site size of 10 Ϯ 1 nucleotides (25). In support of its role as a protein that participates in homologous recombination, it has previously been shown that ICP8 catalyzes reannealing of complementary ssDNA molecules (26).…”