Interaction of herpes primase with the sugar of a NTP

We analyzed the interaction of nucleoside triphosphates (NTPs) containing modified sugars to develop a better understanding of how DNA primase from herpes simplex virus I catalyzes primer synthesis. During the NTP binding reaction, primase tolerated a large number of modifications to the sugar ring. Altering the 2' and 3' carbons and even converting the furanose sugar into an acyclic sugar did not prevent binding. Whether or not the base on the NTP could form a correct base pair with the template base being replicated also had minimal effect on the binding reaction, indicating that primase does not use this process to discriminate between right and wrong NTPs. Rather, the key feature that primase recognizes to bind a NTP is the 5'-gamma-phosphate since converting a NTP into a NDP greatly compromised binding. During the polymerization reaction, primase tolerated substantial modification of the 2'-carbon, including the presence of either an ara or ribo hydroxyl, two hydrogens, or two fluorines. However, polymerization absolutely required that the NTP contain a 3'-hydroxyl and an intact sugar ring. Modifications at the 2'-carbon of the nucleotide at the primer 3'-terminus significantly impaired further polymerization events. Compared to a ribonucleotide, incorporation of a 2'-deoxyribo- or 2',2'-difluoro-2'-deoxyribonucleotide resulted in strong chain termination, while incorporation of an aranucleotide resulted in very strong chain termination. The implications of these data with respect to the mechanism of primase and the relationship between human and herpes primase are discussed.