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J Biol Chem, Vol. 274, Issue 12, 8068-8076, March 19, 1999
From the Department of Microbiology, University of Connecticut
Health Center, Farmington, Connecticut 06030
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.
A Mutation in the C-terminal Putative Zn2+ Finger
Motif of UL52 Severely Affects the Biochemical Activities of
the HSV-1 Helicase-Primase Subcomplex
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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