A sensor complements the steric gate when DNA polymerase ϵ discriminates ribonucleotides

Abstract The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases when building DNA from dNTPs. It is currently believed that DNA polymerases discriminate against NTPs through a steric gate model involving a clash between a tyrosine and the 2′-hydroxyl of the ribonucleotide in the polymerase active site in B-family DNA polymerases. With the help of crystal structures of a B-family polymerase with a UTP or CTP in the active site, molecular dynamics simulations, biochemical assays and yeast genetics, we have identified a mechanism by which the finger domain of the polymerase sense NTPs in the polymerase active site. In contrast to the previously proposed polar filter, our experiments suggest that the amino acid residue in the finger domain senses ribonucleotides by steric hindrance. Furthermore, our results demonstrate that the steric gate in the palm domain and the sensor in the finger domain are both important when discriminating NTPs. Structural comparisons reveal that the sensor residue is conserved among B-family polymerases and we hypothesize that a sensor in the finger domain should be considered in all types of DNA polymerases.


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Supplementary Table 2. Distance (Å) between the Cα-atom of D877 and the OH-group oxygen of Y645 during MD simulations of wild-type and M644G Pol ε in complex with dATP and ATP. a a All distances are average values and standard deviations, averaged over 3 production replicas of 100 ns simulation time each, as described in the Materials and Methods, with snapshots taken every 12.5 ps for the analysis.
Supplementary Table 3. Distance (Å) between the 2'-C atom of the bound nucleotide (dATP/ATP) and the Cδ1 atom of Y645 during MD simulations of wild-type and M644G Pol ε in complex with dATP and ATP. a a All distances are average values and standard deviations, averaged over 3 production replicas of 100 ns simulation time each, as described in the Materials and Methods, with snapshots taken every 12.5 ps for the analysis.
Supplementary Table 4. Angle (°) between the O2B atom of the dNTP and the Cα and Cγ atoms of the N828 side chain during simulations of wild-type and M644G Pol ε in complex with dATP and ATP. a a All distances are average values and standard deviations, averaged over 3 production replicas of 100 ns simulation time each, as described in the Materials and Methods, with snapshots taken every 12.5 ps for the analysis.
Experimental distances for the corresponding atom pairs have been measured from the crystal structure coordinates of wild-type Pol ε (PDB: 4m8o).a a All distances are average values and standard deviations, averaged over 3 production replicas of 100 ns simulation time each, as described in the Materials and Methods, with snapshots taken every 12.5 ps for the analysis.

Kb r0
Zn-D 800.0 0.900 a Rates are relative to the wild-type strain.Measurements were performed as described in the Methods.Values in parentheses are 95% confidence intervals, calculated as described in ref. (4).

Angle type
Supplementary Table 8.Mutations found in the CAN1 gene of Can R yeast strains.

Types of mutations Number of mutations in the different strains
Rates (×10 -8 )

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Supplementary Table 9. Exploring the existence of the possible hydrogen bonds on which the "polar filter" model depends (6).The analysis was performed using the H-bonds tool in ChimeraX (7).a) Here, we separated "strict" hydrogen bonds (fulfilling the precise geometric criteria for a hydrogen bond) from "loose" hydrogen bonds, identified with relaxed hydrogen-bond constraints (by 0.4 Ångströms and 20.0 degrees) indicating that tolerances have been applied to the precise geometric criteria.These standard criteria in ChimeraX came from a survey of high-resolution structures(8); empirically, tolerances of 0.4 Ångström and 20.0 degrees are considered to work well for most macromolecular structures."None" indicates that ChimeraX could not find a hydrogen bond even with the relaxed hydrogen-bond restraints.
Data from Aksenova et al. (5).b 63 out of 74 substitutions were A->T, thus leading to an imbalance that could result from T-T mismatches during leading strand synthesis. a