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

Dry eye stress activates NOTCH1-Dll4 axis and HIF-1α during lymphatic vessel formation of dry eye-induced lacrimal glands.

<p>DE stress activates Dll4/Notch pathway and HIF-1α in LGs. HIF-1α upregulates Dll4/Notch pathway and promotes lymphangiogenesis. Activation of Dll4/Notch pathway and HIF-1α results in increase of VEGF-C, VEGF-D, and VEGFR3, which results in lymphangiogenesis in LGs. (DE = dry eye).</p

Change in NOTCH signaling in dry eye-induced lacrimal glands.

<p>After C57BL/6 mice were housed in a CEC with scopolamine administration for 10 days, LGs were obtained and prepared for qRT-PCR, and (A) mRNA levels of NOTCHs, DLLs, and JAGs were measured. (B) The change in the mRNA level of NOTCH1/DLL4 during DE induction was measured (C) During DE induction, each LG samples were prepared for immunoblot for NOTCH1 and DLL4 at Day 2, Day 4, Day 6, Day 8, and Day 10. Densitometry for protein concentreation quantification was done by using ImageJ software. Student’s t-test for statistical analysis: *p<0.05, **p<0.01, †p<0.001. Error bars indicate standard deviation. (CTL = normal control; DE = dry eye).</p

Change in NOTCH signaling and LYVE-1 expression in dry eye-induced HIF-1α conditional knockout mice.

<p>WT B6 and HIF-1α CKO mice were housed in a CEC with scopolamine administration for 10 days. LGs were obtained and prepared for qRT-PCR and (A) The mRNA level of NOTCH1, DLL4, LYVE-1, and Podoplanin at Day 10 was measured. (B) Immunoblot and densitometry for NOTCH1 and LYVE-1 were measured at Day 10. (C) Immunofluorescence staining of LYVE-1 was performed for DE-induced WT B6 mice and DE-induced HIF-1α CKO mice at Day 10. Student’s t-test for statistical analysis: *p<0.05, **p<0.01. Error bars indicate standard deviation. (WT = wild-type; DE = dry eye; HIF-1α CKO = HIF-1α conditional knockout).</p

Change in LYVE-1, VEGF-C, VEGF-D, and VEGFR3 expression after inhibition of the NOTCH1-Dll4 axis by intraperitoneal injection of monoclonal anti-Dll4 antibody and γ-secretase inhibitor.

<p>While C57BL/6 mice were housed in a CEC with scopolamine administration for 10 days, several groups of mice were administered anti-Dll4 Ab and GSI for inhibiting the NOTCH1-DLL4 axis. Anti-IgG Ab was administered as a control for the anti-Dll4 antibody group, and DMSO was administered as a negative control. LGs were obtained after 10 days of DE induction and were prepared for qRT-PCR, immunoblot, and immunostaining. (A) The mRNA levels of LYVE-1, VEGF-C, VEGF-D, and VEGFR3 were measured using qPCR for each group. (B) Immunoblot and densitometry of LYVE-1 were measured for each group. (C) Immunofluorescence staining of LYVE-1 was performed for each group. Student’s t-test for statistical analysis: *p<0.05, **p<0.01. Error bars indicate standard deviation. (CTL = normal control; DE = dry eye; α-Dll4 = anti-Dll4 antibody; α-IgG = anti-IgG antibody; GSI = γ-secretase inhibitor; DMSO = dissolved dimethyl sulfoxide).</p

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

Change in CD45⁺ cells in dry eye-induced lacrimal glands.

<p>During 10 days of DE induction, (A) LGs were obtained on Days 0, 2, 4, 6, and 10 for IHC staining of CD45⁺ cells. (B) The fold change of CD45⁺ cells was measured at Days 0, 2, 4, 6, and 10. (C) During DE induction, several groups of mice were administered anti-Dll4 Ab and GSI to inhibit the NOTCH1-DLL4 axis. Anti-IgG Ab was administered as a control for the anti-Dll4 Ab group, and DMSO was administered as a negative control. LGs were obtained at Day 10 of DE induction and were prepared for IHC staining of CD45⁺ cells. (D) The actual percentage of CD45⁺ cells was calculated. (E) Flow cytometry for CD45⁺ cells was performed for each condition according to the manufacturer’s protocol. (F) DE was induced for 10 days in B6 and HIF-1CKOmice. LGs were obtained and prepared for IHC staining. (G) The actual percentage of CD45⁺ cells was calculated for non-DE HIF-1α CKO mice and the DE HIF-1α CKO mice. (H) Flow cytometry was performed for the two groups. Student’s t-test for statistical analysis: *p<0.05, **p<0.01, †p<0.001. Error bars indicate standard deviation. (CTL = normal control; DE = dry eye; α-Dll4 = anti-Dll4 antibody; α-IgG = anti-IgG antibody; GSI = γ-secretase inhibitor; DMSO = dissolved dimethyl sulfoxide; HIF-1α CKO = HIF-1α conditional knockout).</p

Change in LYVE-1, PECAM, VEGF-C, VEGF-D, and VEGFR3 expression in dry eye-induced lacrimal glands.

<p>During 10 days of DE induction, LGs were obtained and prepared for qRT-PCR and (A) the mRNA level of LYVE-1 and PECAM was measuredat Day 2, Day 4, Day 6, Day 8, and Day 10 of DE induction. (B) The mRNA levels of VEGF-C, VEGF-D, and VEGFR3 were measured at Day 10. (C) Immunofluorescence staining of LYVE-1 for DE and control group was performed at Day 10. Student’s t-test for statistical analysis: *p<0.05, **p<0.01. Error bars indicate standard deviation. (CTL = normal control; DE = dry eye).</p

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