Biochemical analyses of mutations in the HSV-1 helicase-primase that alter ATP hydrolysis, DNA unwinding, and coupling between hydrolysis and unwinding.
Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase composed of the products of the UL5, UL52, and UL8 genes. UL5 possesses six motifs conserved among superfamily 1 of helicase proteins. Substitutions of conserved residues in each motif abolishes DNA replication in vivo (Zhu, L., and Weller, S. K. (1992) J. Virol. 66, 469-479). Purified UL5.52 harboring a Gly to Ala change in motif V retains primase and helicase activities in vitro but exhibits a higher KM for single-stranded DNA and lower DNA-dependent ATPase activity (Graves-Woodward, K. L., and Weller, S. K. (1996) J. Biol. Chem. 272, 13629-13635). We have purified and characterized six other subcomplexes with residue changes in the UL5 helicase motifs. Each variant subcomplex displays at least wild type or greater levels of primase and DNA binding activities, but all are defective in helicase activity. Mutations in motifs I and II exhibit profound decreases in DNA-dependent ATPase activity. Mutations in motifs III-VI decrease DNA-dependent ATPase activity 3-6-fold. Since mutations in motifs III, IV, V, and VI do not eliminate ATP hydrolysis or DNA binding, we propose that they may be involved in the coupling of these two activities to the process of DNA unwinding. This analysis represents the first comprehensive structure-function analysis of the conserved motifs in helicase superfamily 1.
Graves-Woodward
K L
KL
Department of Microbiology, The University of Connecticut Health Center, Farmington, Connecticut 06030-3205, USA.
Gottlieb
J
J
Challberg
M D
MD
Weller
S K
SK
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D014764:Viral Proteins
D011994:Recombinant Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D004264:DNA Topoisomerases, Type I