Kinetic Approaches to Understanding the Mechanisms of Fidelity of the Herpes Simplex Virus Type 1 DNA Polymerase

Table 2

Summary of presteady-state kinetics of excision by wild-type HSV-1 polymerase.

Enzyme	P/T interface	Fast burst rate constant (sec^-1)	P/T excised at fast rate (%)	Slow burst rate constant (sec^-1)	P/T excised at slow rate (%)
	dA/dT (matched)	59±18^b	6^b	2.2±0.7^b	26^b
WT pol^a	dA/dA (mismatched)	113±7^c	29^c	NA	NA
	dA/AP (abasic)	130±0^c	39^c	NA	NA
WT pol/UL42𝐝	dG/dC (matched)	12±6^b	12.5^b	0.25±0.02^b	87.5^b
	dG (8 nt frayed)	125±7^b	73^b	1.3±0.2^b	27^b
	ACV/dC (matched)	(5.1±0.4)×10^−3c	96^c	NA	NA
	ACV (8 nt frayed)	≥ 0.3^c	100^c	NA	NA
	ACV/dC + dNTP	(2±0.6)×10^−4c	94^c	NA	NA
	ACV (8 nt frayed) + dNTP	≥ 0.3^c	100^c	NA	NA

a The wild-type pol catalytic subunit (25 nM) was incubated in the presence of EDTA with 10 nM of a 46 nt primer annealed to a 67 nt template differing only at the primer/template (P/T) interface as indicated. Single turnover conditions were achieved by initiating reactions with MgCl₂ plus activated calf thymus DNA to trap dissociating pol (from [79]). All kinetic constants are apparent.
bThe amount of full-length primer remaining was plotted as a function of time and the data were fit to the five-parameter double exponential decay function [intact primer] = ae ^-bt + ce ^-dt + 𝑓, where 𝑎 and 𝑏 are the amplitude and burst rate constant, respectively, during the fast phase, and 𝑐 and 𝑑 are the amplitude and burst rate constant during the slow phase of excision. The 𝑓 constant represents the amount of unexcised primer remaining due to dissociation of the enzyme from the P/T or to failure of the enzyme to bind all of the P/T prior to initiation.
cThe amount of full-length primer remaining was plotted as a function of time and the data were fit to a three-parameter single exponential decay function [intact primer] = 𝑎e ^-bt + 𝑐 due to the absence of a slower phase. NA, not applicable for single exponential functions.
dThe wild-type pol/UL42 heterodimer (100 nM) was incubated in the presence of EDTA under single turnover conditions with 90 nM 26 nt primer containing the 3′ nucleotide dGMP (dG) or acyclovir monophosphate (ACV) annealed to a 45 nt template prepared as described [78]. In some cases as indicated, the P/T contained 8 mismatches at the 3′ end of the primer (frayed), the last of which was ACV. When added, the concentration of dNTPs was 100 μM (from [78]). All kinetic constants are apparent.