title: Herpes simplex virus 1 primase employs watson-crick hydrogen bonding to identify cognate nucleoside triphosphates.
abstract: We utilized NTP analogues containing modified bases to probe the mechanism of NTP selection by the primase activity of the herpes simplex virus 1 helicase-primase complex. Primase readily bound NTP analogues of varying base shape, hydrophobicity, and hydrogen-bonding capacity. Remarkably, primase strongly discriminated against incorporating virtually all of the analogues, even though this enzyme misincorporates natural NTPs at frequencies as high as 1 in 7. This included analogues with bases much more hydrophobic than a natural base (e.g., 4- and 7-trifluoromethylbenzimidazole), a base of similar hydrophobicity as a natural base but with the Watson-Crick hydrogen-bonding groups in unusual positions (7-beta-d-guanine), bases shaped almost identically to the natural bases (4-aminobenzimidazole and 4,6-difluorobenzimidazole), bases shaped very differently than a natural base (e.g., 5- and 6-trifluoromethylbenzimidazole), and bases capable of forming just one Watson-Crick hydrogen bond with the template base (purine and 4-aminobenzimidazole). The only analogues that primase readily polymerized into primers (ITP and 3-deaza-ATP) were those capable of forming Watson-Crick hydrogen bonds with the template base. Thus, herpes primase appears to require the formation of Watson-Crick hydrogen bonds in order to efficiently polymerize a NTP. In contrast to primase's narrow specificity for NTP analogues, the DNA-dependent NTPase activity associated with the herpes primase-helicase complex exhibited very little specificity with respect to NTPs containing unnatural bases. The implications of these results with respect to the mechanism of the helicase-primase and current fidelity models are discussed.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Ramirez-Aguilar
authors.forename: Chad L
authors.initials: CL
authors.lastname: Moore
authors.forename: Robert D
authors.initials: RD
authors.lastname: Kuchta
mesh_terms: D000818:Animals; D002460:Cell Line; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019915:DNA Primase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D009711:Nucleotides; D014764:Viral Proteins; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases