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. 2008 Aug 26;47(34):8977-84.
doi: 10.1021/bi8008467. Epub 2008 Aug 2.

Interaction of herpes primase with the sugar of a NTP

Kristopher E Keller  1 , Nisha Cavanaugh, Robert D Kuchta
Affiliations

Interaction of herpes primase with the sugar of a NTP

Kristopher E Keller et al. Biochemistry. .

Abstract

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.

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Figures

Figure 1
Figure 1
Incorporation of C analogues. Panel A. Assays were performed as described under Experimental Procedures and contained primase, d(GTCT)20, ATP, [α-32P]GTP, and 800 μM of the indicated NTP (analogue). Panel B. Assays contained d(GTCT)20, ATP, [α-32P]GTP, and either 0, 100, 200, 400, or 800 μM dFdCTP. In both panels, lanes marked ‘C’ lacked enzyme. Primer lengths are noted on the left.
Figure 2
Figure 2
Incorporation of ddGTP and dGTP. Assays were performed as described under Experimental Procedures and contained primase, d(C20GCCCTA18). ATP. [α-32P]GTP and either 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, or 2 mM of ddGTP (left side) or of dGTP. The lane marked ‘C’ lacked enzyme. Primer lengths are noted on the left.
Figure 3
Figure 3
Incorporation of ATP analogues as the second nucleotide of the primer. Assays were performed as described under Experimental Procedures and contained primase, d(GCTC)15, [α-32P]GTP, and the indicated ATP (analogue). The lane marked ‘C’ lacked enzyme, and the length of products is noted on the left.
Figure 4
Figure 4
Primer elongation with ATP and araATP. Assays were performed as described under Experimental Procedures and contained primase, DNAT and either 0, 0.1, 0.3, 0.5, 0.75, 1 or 2 mM ATP (left side) or araATP (right side). The lane marked ‘C’ lacked enzyme, and the lane marked ‘0’ contained enzyme but no NTP.
Chart 1
Chart 1
Structures and abbreviations of sugars examined.
See this image and copyright information in PMC

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