Identification of the primase active site of the herpes simplex virus type 1 helicase-primase.
Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase composed of the products of the three DNA replication-specific genes UL5, UL8, and UL52 (Crute, J. J., and Lehman, I. R. (1991) J. Biol. Chem. 266, 4484-4488). The UL5 and UL52 products constitute a heterodimeric subassembly of the holoenzyme that contains both helicase and primase activities (Calder, J. M., and Stow, N. D. (1990) Nucleic Acids Res. 18, 3573-3578; Dodson, M. S., and Lehman, I. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1105-1109). The role of the UL52 product in the active HSV-1 helicase-primase was examined. A sequence located between residues 610 and 636 on the UL52 protein was found to be conserved among the UL52 homologues of eight herpesviruses. The carboxyl-terminal portion of this conserved sequence consisted of two Asp residues separated by a variable hydrophobic amino acid residue and is analogous to the divalent metal-binding site of DNA polymerases and several DNA primases. This motif has been designated the herpesvirus primase DXD motif. To study the role of the HSV-1 primase DXD motif in primase action, three site-directed changes were introduced into the UL52 gene. The helicase activity of the recombinant holoenzymes was unaffected by any of the introduced changes. Changing either of the two Asp residues that constitute the divalent metal-binding site (Asp628 or Asp630) to Ala dramatically reduced the primase activity of the HSV-1 helicase-primase holoenzyme in vitro, whereas alteration of the nearby conserved residue Asn624 to Gly had minimal effect. Therefore, in the three-subunit HSV-1 helicase-primase, the UL52 product provides at least a part of the primase catalytic site.
Dracheva
S
S
Department of Immunological Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368, USA.
Koonin
E V
EV
Crute
J J
JJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis; D004265:DNA Helicases / Q000737:chemistry* / Q000502:physiology; D019915:DNA Primase; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D008969:Molecular Sequence Data; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000502:physiology
D004247:DNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases