Effects of <i>N</i>²‑Alkylguanine, <i>O</i>⁶‑Alkylguanine, and Abasic Lesions on DNA Binding and Bypass Synthesis by the Euryarchaeal B‑Family DNA Polymerase Vent (exo^–)

Archaeal and eukaryotic B-family DNA polymerases (pols) mainly replicate chromosomal DNA but stall at lesions, which are often bypassed with Y-family pols. In this study, a B-family pol Vent (exo^–) from the euryarchaeon <i>Thermococcus litoralis</i> was studied with three types of DNA lesions<i>N</i>²-alkylG, <i>O</i>⁶-alkylG, and an abasic (AP) sitein comparison with a model Y-family pol Dpo4 from <i>Sulfolobus solfataricus</i>, to better understand the effects of various DNA modifications on binding, bypass efficiency, and fidelity of pols. Vent (exo^–) readily bypassed <i>N</i>²-methyl­(Me)­G and <i>O</i>⁶-MeG, but was strongly blocked at <i>O</i>⁶-benzyl­(Bz)­G and <i>N</i>²-BzG, whereas Dpo4 efficiently bypassed <i>N</i>²-MeG and <i>N</i>²-BzG and partially bypassed <i>O</i>⁶-MeG and <i>O</i>⁶-BzG. Vent (exo^–) bypassed an AP site to an extent greater than Dpo4, corresponding with steady-state kinetic data. Vent (exo^–) showed ∼110-, 180-, and 300-fold decreases in catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) for nucleotide insertion opposite an AP site, <i>N</i>²-MeG, and <i>O</i>⁶-MeG but ∼1800- and 5000-fold decreases opposite <i>O</i>⁶-BzG and <i>N</i>²-BzG, respectively, as compared to G, whereas Dpo4 showed little or only ∼13-fold decreases opposite <i>N</i>²-MeG and <i>N</i>²-BzG but ∼260–370-fold decreases opposite <i>O</i>⁶-MeG, <i>O</i>⁶-BzG, and the AP site. Vent (exo^–) preferentially misinserted G opposite <i>N</i>²-MeG, T opposite <i>O</i>⁶-MeG, and A opposite an AP site and <i>N</i>²-BzG, while Dpo4 favored correct C insertion opposite those lesions. Vent (exo^–) and Dpo4 both bound modified DNAs with affinities similar to unmodified DNA. Our results indicate that Vent (exo^–) is as or more efficient as Dpo4 in synthesis opposite <i>O</i>⁶-MeG and AP lesions, whereas Dpo4 is much or more efficient opposite (only) <i>N</i>²-alkylGs than Vent (exo^–), irrespective of DNA-binding affinity. Our data also suggest that Vent (exo^–) accepts nonbulky DNA lesions (e.g., <i>N</i>²- or <i>O</i>⁶-MeG and an AP site) as manageable substrates despite causing error-prone synthesis, whereas Dpo4 strongly favors minor-groove <i>N</i>²-alkylG lesions over major-groove or noninstructive lesions

Biochemical Analysis of Six Genetic Variants of Error-Prone Human DNA Polymerase ι Involved in Translesion DNA Synthesis

DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., <i>N</i>²-ethyl­(Et)­G, <i>O</i>⁶-methyl­(Me)­G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1–445) proteins and DNA templates containing a G, <i>N</i>²-EtG, <i>O</i>⁶-MeG, 8-oxoG, or abasic site. The Δ1–25 variant, which is the <i>N</i>-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg²⁺ (but not with Mn²⁺), coinciding with its steady-state kinetic data showing a ∼10-fold increase in <i>k</i>_cat/<i>K</i>_m for nucleotide incorporation opposite templates (only with Mg²⁺). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in <i>k</i>_cat/<i>K</i>_m for nucleotide incorporation opposite templates either with Mg²⁺ or Mn²⁺, except for that opposite <i>N</i>²-EtG with Mn²⁺ (showing a 9-fold increase for dCTP incorporation). The Δ1–25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg²⁺), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1–25 variant, was ∼7-fold stronger with 0.15 mM Mn²⁺ than with Mg²⁺. The results indicate that the R96G variation severely impairs most of the Mg²⁺- and Mn²⁺-dependent TLS abilities of pol ι, whereas the Δ1–25 variation selectively and substantially enhances the Mg²⁺-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences in TLS, mutation, and cancer susceptibility to genotoxic carcinogens

Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky <i>N</i>²‑Guanyl DNA Adducts

DNA polymerase (pol) κ, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky <i>N</i>²-guanyl DNA lesions induced by many carcinogens such as polycyclic aromatic hydrocarbons. We analyzed the biochemical properties of eight reported human pol κ variants positioned in the polymerase core domain, using the recombinant pol κ (residues 1–526) protein and the DNA template containing an <i>N</i>²-CH₂(9-anthracenyl)­G (<i>N</i>²-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol κ. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in <i>k</i>_cat/<i>K</i>_m for dCTP insertion opposite G and <i>N</i>²-AnthG compared to that of wild-type pol κ. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in <i>k</i>_cat/<i>K</i>_m for next-base extension from C paired with <i>N</i>²-AnthG, compared to that of wild-type pol κ. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol κ opposite bulky <i>N</i>²-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens

Leukotriene Biosynthesis Inhibitor MK886 Impedes DNA Polymerase Activity

Specialized DNA polymerases participate in replication stress responses and in DNA repair pathways that function as barriers against cellular senescence and genomic instability. These events can be co-opted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments and as such, represent potential targets for adjuvant therapies. Previously, a high-throughput screen of ∼16,000 compounds identified several first generation proof-of-principle inhibitors of human DNA polymerase kappa (hpol κ). The indole-derived inhibitor of 5-lipoxygenase activating protein (FLAP), MK886, was one of the most potent inhibitors of hpol κ discovered in that screen. However, the specificity and mechanism of inhibition remained largely undefined. In the current study, the specificity of MK886 against human Y-family DNA polymerases and a model B-family DNA polymerase was investigated. MK886 was found to inhibit the activity of all DNA polymerases tested with similar IC₅₀ values, the exception being a 6- to 8-fold increase in the potency of inhibition against human DNA polymerase iota (hpol ι), a highly error-prone enzyme that uses Hoogsteen base-pairing modes during catalysis. The specificity against hpol ι was partially abrogated by inclusion of the recently annotated 25 a.a. N-terminal extension. On the basis of Michaelis–Menten kinetic analyses and DNA binding assays, the mechanism of inhibition by MK886 appears to be mixed. <i>In silico</i> docking studies were used to produce a series of models for MK886 binding to Y-family members. The docking results indicate that two binding pockets are conserved between Y-family polymerases, while a third pocket near the thumb domain appears to be unique to hpol ι. Overall, these results provide insight into the general mechanism of DNA polymerase inhibition by MK886

Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-Free Bypass across Bulky <i>N</i>²‑Guanyl DNA Adducts

DNA polymerase (pol) κ, one of the Y-family polymerases, has been shown to function in error-free translesion DNA synthesis (TLS) opposite the bulky <i>N</i>²-guanyl DNA lesions induced by many carcinogens such as polycyclic aromatic hydrocarbons. We analyzed the biochemical properties of eight reported human pol κ variants positioned in the polymerase core domain, using the recombinant pol κ (residues 1–526) protein and the DNA template containing an <i>N</i>²-CH₂(9-anthracenyl)­G (<i>N</i>²-AnthG). The truncation R219X was devoid of polymerase activity, and the E419G and Y432S variants showed much lower polymerase activity than wild-type pol κ. In steady-state kinetic analyses, E419G and Y432S displayed 20- to 34-fold decreases in <i>k</i>_cat/<i>K</i>_m for dCTP insertion opposite G and <i>N</i>²-AnthG compared to that of wild-type pol κ. The L21F, I39T, and D189G variants, as well as E419G and Y432S, displayed 6- to 22-fold decreases in <i>k</i>_cat/<i>K</i>_m for next-base extension from C paired with <i>N</i>²-AnthG, compared to that of wild-type pol κ. The defective Y432S variant had 4- to 5-fold lower DNA-binding affinity than wild-type, while a slightly more efficient S423R variant possessed 2- to 3-fold higher DNA-binding affinity. These results suggest that R219X abolishes and the E419G, Y432S, L21F, I39T, and D189G variations substantially impair the TLS ability of pol κ opposite bulky <i>N</i>²-G lesions in the insertion step opposite the lesion and/or the subsequent extension step, raising the possibility that certain nonsynonymous pol κ genetic variations translate into individual differences in susceptibility to genotoxic carcinogens