Abstract
Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex that is composed of the products of the UL5, UL52, and UL8 genes. A subcomplex consisting of the UL5 and UL52 proteins retains all the enzymatic activities exhibited by the holoenzyme in vitro. The UL52 protein contains a putative zinc finger at its C terminus which is highly conserved among both prokaryotic and eukaryotic primases. We constructed a mutation in which two highly conserved cysteine residues in the zinc finger motif were replaced with alanine residues. A UL52 expression plasmid containing the mutation in the zinc finger region is unable to support the growth of a UL52 mutant virus in a transient complementation assay. Wild type and mutant UL5.UL52 subcomplexes were purified from insect cells infected with recombinant baculoviruses. Surprisingly, the mutant protein was severely affected in all biochemical activities tested; no helicase or primase activities could be detected, and the mutant protein retains only about 9% of wild type levels of single-stranded DNA-dependent ATPase activity. Gel mobility shift assays showed that DNA binding is severely affected as well; the mutant subcomplex only retains approximately 8% of wild type levels of binding to a forked substrate. On the other hand, the mutant protein retains its ability to interact with UL5 as indicated by copurification and with UL8 as indicated by a supershifted band in the gel mobility shift assay. In addition, the ability of individual subunits to bind single-stranded DNA was examined by photo cross-linking. In the wild type UL5.UL52 subcomplex, both subunits are able to bind an 18-mer of oligo(dT). The mutant subcomplex was severely compromised in the ability of both UL5 and UL52 to bind the oligonucleotide; total cross-linking was only 2% of wild type levels. These results are consistent with the proposal that the putative zinc binding motif of UL52 is required not only for binding of the UL52 subunit to DNA and for primase activity but also for optimal binding of UL5 to DNA and for the subsequent ATPase and helicase activities.
Highlights
Herpes simplex virus type 1 (HSV-1)1 encodes a heterotrimeric DNA helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes (1, 2)
The HSV-1 helicase-primase complex can be isolated from insect cells that have been simultaneously infected with recombinant baculoviruses that express each of the three subunits (8)
Site-directed Mutagenesis of the Zinc Finger Motif of UL52— Site-directed mutagenesis was used to explore the functional significance of the putative zinc finger region of the UL52 protein
Summary
Supplemented Graces’s medium, 10% Pluronic®F-68 and the Bac-toBacTM recombinant baculovirus kit were purchased from Life Technologies, Inc. Fetal calf serum was obtained from Atlanta Biologicals. Penicillin/streptomycin solution, ampicillin, phenylmethylsulfonyl fluoride, leupeptin, and pepstatin were purchased from Sigma. The 20-ml HiLoad 16/10 SP Sepharose Fast Flow column was from Amersham Pharmacia Biotech. The 12-ml Uno Q (Q-12) column was from Bio-Rad. Radiolabeled nucleotides were purchased from Amersham Pharmacia Biotech. Oligonucleotides were synthesized by Life Technologies, Inc. The oligonucleotide substituted with 5-iodo deoxyuridine was synthesized by Cruachem (Dulles, VA). Buffer A consists of 20 mM HEPES, pH 7.6, 1.0 mM dithiothreitol (DTT), 10 mM sodium bisulfite, 5 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 g/ml leupeptin, 1 g/ml pepstatin, and 2 g/ml aprotinin. Buffer B contains 20 mM HEPES, pH 7.6, 1.0 mM DTT, 10% (V/V) glycerol, and 0.5 mM EDTA. All buffers were passed through a 0.22-m filter and degassed before use
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