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 ε (Polε) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polε 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, Polε 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 Polε heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polε in vitro. However, similar studies of the human Polε heterote-tramer (hPolε) have been limited by the difficulty of obtaining hPolε in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolε from insect host cells has allowed for isolation of greater amounts of active hPolε, thus enabling a more detailed kinetic comparison between hPolε and an active N-terminal fragment of the hPolε catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolε. We observe that the small subunits increase DNA binding by hPolε relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3′ → 5′ exonuclease activity of hPolε 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 hPolε and sway hPolε toward DNA synthesis rather than proofreading.

Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair

“…Using an in vitro lacZ reversion substrate that specifically measures TCT→TAT transversions ( Shinbrot et al, 2014 ; Shlien et al, 2015 ), the D275A/E277A mutant made these errors at a significantly higher rate in vitro than the wild type exonuclease-proficient Pol ε enzyme ( Figure 1C ). We used a construct comprised of the N-terminal 140 kDa of Pol ε, which contains the DNA polymerase and exonuclease domains and has similar fidelity and catalytic activity to the complete four subunit holoenzyme ( Aksenova et al, 2010 ; Ganai et al, 2015 ; Zahurancik et al, 2015 ). Importantly, the elevated TCT→TAT error rate we observed with the D275A/E277A mutant was not statistically different from those measured with the S459F and S461P Pol ε cancer mutants previously ( Shinbrot et al, 2014 ; Shlien et al, 2015 ), suggesting a common mechanism of mutagenesis for these hotspot mutations.…”

Crystal structure of the human Polϵ B-subunit in complex with the C-terminal domain of the catalytic subunit

“…Thus, all structural data support our previous conclusion (25) that Pol⑀ and Pol␣ require only zinc for correct assembly (5, 44). Moreover, concentrated and very pure samples of human Pol␣ and Pol⑀, obtained in our laboratory, were ironfree and fully functional (46,47), indicating that their operation in eukaryotic cells is not dependent on the iron-sulfur cluster assembly machinery.…”

Kinetic investigation of the polymerase and exonuclease activities of human DNA polymerase ε holoenzyme

“…In a study that followed, we examined wildtype hPolε holoenzyme prepared from baculovirus-infected insect cells and performed burst assays with both hPolε and p261N under identical conditions. We observed that both exhibited a fast burst phase and a slow linear phase of product formation (23). The multiphasic kinetics of correct nucleotide incorporation suggests that hPolε likely utilizes a similar minimal kinetic mechanism as p261N (Scheme 1A) for DNA polymerization.…”

“…Specifically, a fixed concentration of hPolε exo-(50 nM, UV concentration) was pre-incubated with varying concentrations of D-1 DNA substrate (10-125 nM) to allow the E•DNA complex to form prior to initiation of the reaction with the addition of correct dTTP (100 μM) and Mg 2+ . The concentration of the product formed during the first turnover was measured by quenching each reaction after 50 ms which allowed adequate time for the dTTP incorporation to reach the maximum first-turnover amplitude with a negligible contribution of multiple turnovers (23). Importantly, the amplitude of product formation from the first turnover was a direct measurement of the amount of productive E•DNA complex that formed during the pre-incubation period.…”

“…We then determined that the multipleturnover (or steady-state) rate (0.007 s -1 ) of nucleotide incorporation was governed by the hPolε•DNA complex dissociation (0.0058 s -1 ) (Figure 1). Through an active site titration assay, the apparent binding affinity of D-1 DNA substrate to hPolε (1/Kd,app DNA , Kd,app DNA = 22 nM) was measured (Figure 2), indicating that the first turnover of nucleotide incorporation was much faster than the equilibration of hPolε and DNA binding (E + DNA  E•DNA) (23).…”