Herpes Simplex Virus Processivity Factor UL42 Imparts Increased DNA-Binding Specificity to the Viral DNA Polymerase and Decreased Dissociation from Primer-Template without Reducing the Elongation Rate

Abstract

Herpes simplex virus DNA polymerase consists of a catalytic subunit, Pol, and a processivity subunit, UL42, that, unlike other established processivity factors, binds DNA directly. We used gel retardation and filter-binding assays to investigate how UL42 affects the polymerase-DNA interaction. The Pol/UL42 heterodimer bound more tightly to DNA in a primer-template configuration than to single-stranded DNA (ssDNA), while Pol alone bound more tightly to ssDNA than to DNA in a primer-template configuration. The affinity of Pol/UL42 for ssDNA was reduced severalfold relative to that of Pol, while the affinity of Pol/UL42 for primer-template DNA was increased ∼15-fold relative to that of Pol. The affinity of Pol/UL42 for circular double-stranded DNA (dsDNA) was reduced drastically relative to that of UL42, but the affinity of Pol/UL42 for short primer-templates was increased modestly relative to that of UL42. Pol/UL42 associated with primer-template DNA ∼2-fold faster than did Pol and dissociated ∼10-fold more slowly, resulting in a half-life of 2 h and a subnanomolar Kd. Despite such stable binding, rapid-quench analysis revealed that the rates of elongation of Pol/UL42 and Pol were essentially the same, ∼30 nucleotides/s. Taken together, these studies indicate that (i) Pol/UL42 is more likely than its subunits to associate with DNA in a primer-template configuration rather than nonspecifically to either ssDNA or dsDNA, and (ii) UL42 reduces the rate of dissociation from primer-template DNA but not the rate of elongation. Two models of polymerase-DNA interactions during replication that may explain these findings are presented.

HSV-1 DNA Replication—Coordinated Regulation by Viral and Cellular Factors

“…UL30 has polymerase activity to extend DNA and 3’–5’ exonuclease activity to remove mismatched nucleotides [ 55 , 56 ]. UL30 has an approximate elongation rate of 30 nucleotides per second [ 15 , 57 , 58 ]. The slow elongation rate may help increase polymerase fidelity so that mis-incorporated nucleotides can be excised by the 3’–5’ exonuclease activity.…”

“…The intrinsic ability of UL42 to bind to DNA is crucial for HSV-1 replication [ 65 ]. UL42 functions to prevent the premature dissociation of UL30 from DNA by decreasing the dissociation rate of UL30 by 10-fold, while having no effect on the association rate of UL30 with viral DNA [ 58 ].…”