N. Biswas et Sk. Weller, The UL5 and UL52 subunits of the herpes simplex virus type 1 helicase-primase subcomplex exhibit a complex interdependence for DNA binding, J BIOL CHEM, 276(20), 2001, pp. 17610-17619
Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase compl
ex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protei
n 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 o
f wild type and mutant subcomplexes were examined using single-stranded, du
plex, 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) stimula
ted the binding to a forked substrate. We have previously shown that both s
ubunits contact single-stranded DNA in a photocrosslinking assay (Biswas, N
., and Weller, S, K. (1999) J, Biol Chem 274, 8068-8076). In this study, ph
otocross-linking assays with forked substrates indicate that the UL5 and UL
52 subunits contact the forked substrates at different positions, UL52 at t
he single-stranded DNA tail and UL5 near the junction between single-strand
ed and double-stranded DNA. Neither subunit was able to cross-link a forked
substrate when B-iododeoxyuridine was located within the duplex portion. P
hotocross-linking experiments with subcomplexes containing mutant versions
of UL5 and wild type UL52 indicated that the integrity of the ATP binding r
egion is important for DNA binding of both subunits, These results support
our previous proposal that UL5 and UL52 exhibit a complex interdependence f
or 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 he
licase activity than had previously been thought.