Polymerase and Exonuclease Activities in Herpes Simplex Virus Type 1 DNA Polymerase Are Not Highly Coordinated

Abstract

The herpes polymerase–processivity factor complex consists of the catalytic UL30 subunit containing both polymerase and proofreading exonuclease activities and the UL42 subunit that acts as a processivity factor. Curiously, the highly active exonuclease has minimal impact on the accumulation of mismatches generated by the polymerase activity. We utilized a series of oligonucleotides of defined sequence to define the interactions between the polymerase and exonuclease active sites. Exonuclease activity requires unwinding of two nucleotides of the duplex primer–template. Surprisingly, even though the exonuclease rate is much higher than the rate of DNA dissociation, the exonuclease degrades both single- and double-stranded DNA in a nonprocessive manner. Efficient proofreading of incorrect nucleotides incorporated by the polymerase would seem to require efficient translocation of DNA between the exonuclease and polymerase active sites. However, we found that translocation of DNA from the exonuclease to polymerase active site is remarkably inefficient. Consistent with inefficient translocation, the DNA binding sites for the exonuclease and polymerase active sites appear to be largely independent, such that the two activities appear noncoordinated. Finally, the presence or absence of UL42 did not impact the coordination of the polymerase and exonuclease activities. In addition to providing fundamental insights into how the polymerase and exonuclease function together, these activities provide a rationale for understanding why the exonuclease minimally impacts accumulation of mismatches by the purified polymerase and raise the question of how these two activities function together in vivo.

Different Divalent Cations Alter the Kinetics and Fidelity of DNA Polymerases

“…Similar to the polymerase active site, the exonuclease site also requires divalent cations to catalyze the 3Ј-5Ј-exonuclease activity associated with several DNA pols (5,30,32,35,54,55). Divalent cations are required but have a varying effect on the exonuclease activity.…”

“…All DNA polymerases (DNA pols) 2 require Mg 2ϩ or Mn 2ϩ for primer extension and for excision of incorrectly incorporated dNTPs via intrinsic 3Ј35Ј exonuclease activity (1)(2)(3)(4)(5). A two-metal-ion mechanism is used by all DNA pols to catalyze nucleotide addition to a growing primer strand (6).…”

Effects of Acyclovir, Foscarnet, and Ribonucleotides on Herpes Simplex Virus-1 DNA Polymerase: Mechanistic Insights and a Novel Mechanism for Preventing Stable Incorporation of Ribonucleotides into DNA

“…We previously showed that formation of an E-DNA-aphidicolin ternary complex where the DNA was bound in the polymerase active site had no effect on the exonuclease activity (30), raising the possibility that other inhibitors of herpes polymerase might likewise generate complexes with inhibited polymerase activity but active exonuclease. To test this possibility, we examined the effects of two clinically useful anti-herpes drugs, acyclovir and phosphonoformic acid, on exonuclease activity using synthetic oligonucleotides of defined sequence.…”

“…We previously showed that under conditions of excess DNA over UL30 (or the UL30/UL42 complex), the enzyme processes some of the DNA via dNTP polymerization and some via exonuclease activity (30). Thus, these conditions allow simultaneous monitoring of both polymerase and exonuclease activity.…”

“…We recently showed that blocking the polymerase active site via formation of an UL30-DNA-Aphidicolin dead-end complex has little or no effect on the exonuclease activity, indicating that the polymerase and exonuclease active sites have independent DNA binding domains (30). This result also raises the possibility that other inhibitors may block polymerase activity but leave the exonuclease unbothered.…”