Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ɛ

Abstract

Numerous genetic studies have provided compelling evidence to establish DNA polymerase ɛ (Polɛ) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polɛ is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3'→5' exonuclease domain common to many replicative polymerases. In addition, Polɛ possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polɛ heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polɛ in vitro. However, similar studies of the human Polɛ heterotetramer (hPolɛ) have been limited by the difficulty of obtaining hPolɛ in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolɛ from insect host cells has allowed for isolation of greater amounts of active hPolɛ, thus enabling a more detailed kinetic comparison between hPolɛ and an active N-terminal fragment of the hPolɛ catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolɛ. We observe that the small subunits increase DNA binding by hPolɛ relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3'→5' exonuclease activity of hPolɛ is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolɛ and sway hPolɛ toward DNA synthesis rather than proofreading.

Keywords: 3′→5′ exonuclease activity; DNA polymerase epsilon; Human DNA replication; Leading strand replication; Pre-steady-state kinetics.

Fig. 1

Analysis of hPolε purity by SDS-PAGE. To evaluate the purity of hPolε, the final protein sample was run on a 12% SDS-PAGE gel: lane 1, protein marker; lane 2, eluate from Superose 12 size-exclusion column. The p12 and p17 subunits have identical mobilities and are indistinguishable.

Fig. 2

Biphasic kinetics of correct dTTP incorporation by hPolε and p261N at 20 °C. A pre-incubated solution of 10 nM hPolε (●) or p261N (■) and 40 nM 5′-radiolabeled D-1 DNA was rapidly mixed with 5 μM dTTP and 8 mM Mg²⁺ and quenched after various times with 0.37 M EDTA. The data were fit to Eq. (1) to yield burst phase rate constants of 90 ± 28 s⁻¹ and 101 ± 14 s⁻¹ and linear phase rate constants of 0.047 ± 0.006 s⁻¹ and 0.018 ± 0.004 s⁻¹ for hPolε and p261N, respectively.

Fig. 3

Active site titration of hPolε at 20 °C. A pre-incubated solution of hPolε (50 nM) and increasing concentrations of 5′-radiolabeled D-1 DNA (10–80 nM) was rapidly mixed with dTTP (5 μM). All reactions were quenched after 100 ms with the addition of 0.37 M EDTA. The data were fit to Eq. (2) to yield a $K_{\mathrm{d}}^{\text{DNA}}$ of 33 ± 5 nM and _d an enzyme active concentration of 9.0 ± 0.7 nM.

Fig. 4

Processive DNA synthesis by hPolε and p261N on singly-primed M13mp2 ssDNA templates at 20 °C. A pre-incubated solution of hPolε or p261N (250 nM) and 5′-radiolabeled (A) 21- or (B) 45-mer primer annealed to M13 ssDNA (25 nM) was mixed with all four dNTPs (100 μM) for various times before quenching with the addition of 0.37 M EDTA. Products extended from the 21-mer primer were separated by 17% denaturing PAGE, while products extended from the 45-mer primer were separated by 8% denaturing PAGE.

Fig. 5

Excision of matched and mismatched DNA by hPolε at 20 °C. A pre-incubated solution of 100 nM hPolε (●) or p261N (■) and 20 nM 5′-radiolabeled (A) D-1 or (B) M-1 DNA was rapidly mixed with Mg²⁺ and quenched after various times with 0.37 M EDTA. The data were fit to Eq. (3) to yield k_exo. For the matched D-1 DNA (A), the measured k_exo values were 0.018 ± 0.002 s⁻¹ and 0.041 ± 0.004 s⁻¹ for hPolε and p261N, respectively. For the mismatched M-1 DNA (B), the measured k_exo values were 0.19 ± 0.03 s⁻¹ and 1.4 ± 0.2 s⁻¹ for hPolε and p261N, respectively.