The UL5, UL8, and UL52 genes of herpes simplex virus type 1 encode a multisubunit assembly that possesses primase, DNA helicase, and DNA-dependent nucleoside triphosphatase activities. A subassembly consisting of the UL5 and UL52 gene products retains these activities. The nucleoside triphosphatase activity of the UL5/UL52 subassembly is strongly stimulated by both homo-and heteropolymeric single-stranded DNA. Double-stranded DNA has little ability to stimulate the ATPase activity. The subassembly binds both double and single-stranded DNA. Nucleotides are not required for DNA-binding. The minimum length of single-stranded DNA that is bound and that stimulates enzymatic activity is about 12 nucleotides. The kinetic parameters of the ATPase activity of the subassembly are affected by the length of the oligonucleotide coeffector. The K m decreases as the coeffector length is increased up to a length of about 20 nucleotides and then remains independent of coeffector length. The first order rate constant for ATPase activity exhibits a quasihyperbolic dependence on the length of the DNA coeffector and is maximal for coeffectors of 20 nucleotides and longer.Herpes simplex virus type 1 (HSV-1) 1 encodes a heterotrimeric DNA helicase-primase whose subunits are encoded by the UL5, UL8, and UL52 genes of the virus (1, 2). The helicase activity of the enzyme is coupled to the hydrolysis of either ATP or GTP (1, 3). The UL5 gene product possesses a set of domains that correspond with several conserved motifs found in other DNA helicases (4). Mutagenesis of any one of these conserved domains in UL5 protein obliterates viral DNA synthesis (5). The UL52 gene is also essential for viral DNA synthesis and encodes a conserved domain that is found in other primases (6 -8). Mutagenesis of this conserved domain specifically obliterates the primase activity of the HSV-1 helicase-primase (7,8). Deletion of the UL8 gene from HSV-1 renders the virus incapable of DNA replication (9).The HSV-1 helicase-primase can be isolated from insect cells that have been simultaneously infected with recombinant baculoviruses that express each of the three subunits that compose the holoenzyme (10). A subassembly consisting of the UL5 and UL52 gene products also exhibits the DNA-dependent NTPase, DNA helicase, and primase activities that are associated with the holoenzyme (11, 12). The primase activity of this subassembly exhibits DNA sequence dependence and is stimulated by the UL8 protein (13-15). However, purified UL8 protein itself lacks any type of discernible enzymatic activity (11,12).Homologs of the genes encoding the HSV-1 helicase-primase are found in several other human herpesviruses (16). The complex array of activities possessed by the helicase-primases encoded by this group of viruses may be attractive targets for antiviral drug design. An understanding of the properties of the HSV-1 helicase-primase may lead to strategies for developing such compounds.In this work we further examine the interaction of the UL5/ UL52 subassembly with various...