Herpes simplex virus 1 primase employs watson-crick hydrogen bonding to identify cognate nucleoside triphosphates

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-β-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 (pu...