Abstract
Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains several conserved motifs found in primases; however, the contributions of each subunit to the biochemical activities of the subcomplex are not clear. In this work, the DNA binding properties of wild type and mutant subcomplexes were examined using single-stranded, duplex, and forked substrates. A gel mobility shift assay indicated that the UL5-UL52 subcomplex binds more efficiently to the forked substrate than to either single strand or duplex DNA. Although nucleotides are not absolutely required for DNA binding, ADP stimulated the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and adenosine 5'-O-(thiotriphosphate) stimulated the binding to a forked substrate. We have previously shown that both subunits contact single-stranded DNA in a photocross-linking assay (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked substrate when 5-iododeoxyuridine was located within the duplex portion. Photocross-linking experiments with subcomplexes containing mutant versions of UL5 and wild type UL52 indicated that the integrity of the ATP binding region is important for DNA binding of both subunits. These results support our previous proposal that UL5 and UL52 exhibit a complex interdependence for DNA binding (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active role in helicase activity than had previously been thought.
Highlights
To form ssDNA using the energy of NTP hydrolysis
To determine whether nucleotide di- and triphosphates play a role in the DNA binding properties of UL5-UL52, the binding of the subcomplex with ss and forked DNA substrates was tested in the presence of ATP, ADP, and ATP␥S (Fig. 1)
The similar levels of stimulation observed with ADP, ATP, and ATP␥S suggest that the binding of ATP but not its hydrolysis is important for optimal binding of UL5-UL52 to the forked substrate
Summary
Reagents—Supplemented Graces’s medium and 10% Pluronic®F were purchased from Life Technologies, Inc. Protein Expression and Purification—2 liters of Sf9 cells were grown in suspension at 27 °C in Graces’s insect medium as described previously [39]. UL5-UL52 subcomplexes were precipitated from the cytosolic extract by the addition of an equal volume of Buffer B containing 0.2 M NaCl and 2 M ammonium sulfate and incubation on ice for 4 h. The products were subjected to electrophoresis on an 8% nondenaturing polyacrylamide gel, and the forked substrate was purified by electroelution and ethanol precipitation. The reaction mixture (25 l) contained 20 mM Naϩ HEPES (pH 7.6), 1 mM DTT, 0.1 mg/ml bovine serum albumin, 10% glycerol, 5 mM MgCl2, 1.2 pmol (molecules) of the DNA substrates labeled with [␥-P32]ATP and 4 pmol of the UL5-UL52 subcomplex with or without UL8 protein (12 pmol), ATP (5 mM), ADP (5 mM), or ATP␥S (5 mM). The gels were dried and exposed to film at Ϫ70 °C
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