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Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η*

  • Yan Su
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Martin Egli
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • F.Pete. Guengerich
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University School of Medicine, 638B Robinson Research Bldg., 2200 Pierce Ave., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-343-0704; E-mail:
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01 ES010375 (to F. P. G. and M. E.), R01 ES010546 (to F. P. G.), and P01 CA160032 (to M. E.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Open AccessPublished:January 06, 2016DOI:https://doi.org/10.1074/jbc.M115.706226
      Ribonucleotides and 2′-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2′-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2′-OH group. Y-family human DNA polymerase η (hpol η) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2′-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 103-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol η scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2′-deoxyguanosine with a significant propeller twist. As a result, the 2′-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion.

      Introduction

      RNA and DNA are fundamental to life in all forms. The two nucleic acid polymers are composed of ribonucleotides and 2′-deoxyribonucleotides as the basic units, respectively. However, ribonucleotides have been found in DNA; they constitute a large proportion of the “DNA lesions” in the genome. In mice, they have been shown to be collectively the most frequently occurring DNA lesions, even more than abasic sites and 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG).
      The abbreviations used are: 8-oxodG, 7,8-dihydro-8-oxo-2′-deoxyguanosine; h, human; pol, DNA polymerase; CPD, cyclobutane pyrimidine dimer; PDB, Protein Data Bank; dAMPNPP, 2′-deoxyadenosine-5′-[(α,β)-imido]triphosphate; dCMPNPP, 2′-deoxycytidine-5′-[(α,β)-imido]triphosphate.
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      ). Y-family human DNA polymerase η (hpol η) is of particular interest, as it is the only known DNA polymerase directly related to a human genetic disorder, mainly due to its unique role in translesion synthesis past the UV-induced DNA lesion cyclobutane pyrimidine dimer (CPD). Patients defective in hpol η, the result of which is a form of xeroderma pigmentosum (XP-V), are typically highly sensitive to UV light, with increased incidence of skin and other types of cancer and (for some of the individuals) neurodegeneration (
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      In this study, we investigated the ability of hpol η to incorporate ribonucleotides into DNA and crystallized hpol η with incoming rNTPs opposite both undamaged DNA and an 8-oxodG lesion. Our results demonstrate that hpol η can incorporate ribonucleotides into DNA with relatively high selectivity but low efficiency, even when the template strand contains the DNA lesion 8-oxodG or CPD. The x-ray crystal structures show that the incoming rNTP at the hpol η active site adopts a slightly different orientation relative to dNTP to avoid a clash with the steric gate residue, without significantly disrupting the pairing with the template dG or 8-oxodG. The latter appears to be the first crystal structure of an incoming rNTP opposite a DNA lesion within a DNA polymerase.

      Experimental Procedures

      Materials

      Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA) or TriLink BioTechnologies (San Diego) and purified by HPLC by the manufacturers. rNTPs and dNTPs were purchased from New England Biolabs (Ipswich, MA). These experiments were conducted with the catalytic core of hpol η or the Y92A mutant (1–432 amino acids), and the former has been shown to have similar catalytic activity as the full-length protein in vitro (
      • Zhao Y.
      • Biertümpfel C.
      • Gregory M.T.
      • Hua Y.J.
      • Hanaoka F.
      • Yang W.
      Structural basis of human DNA polymerase η-mediated chemoresistance to cisplatin.
      ). The hpol η and the Y92A mutant (1–432 amino acids) were expressed and purified as reported previously (
      • Su Y.
      • Patra A.
      • Harp J.M.
      • Egli M.
      • Guengerich F.P.
      Roles of residues Arg-61 and Gln-38 of human DNA polymerase η in bypass of deoxyguanosine and 7,8-dihydro-8-oxo-2′-deoxyguanosine.
      ). Polyethylene glycol monomethyl ether 2000 (from Hampton Research, Aliso Viejo, CA) was used for crystallization.

      DNA Substrates

      The fluorescently labeled primer 5′-6-carboxyfluorescein-CGG GCT CGT AAG CGT CAT-3′ was annealed with each of the following template oligonucleotides at a 1:1 molar ratio by heating to 95 °C and slowly cooling: 1) 5′-TCA TGA TGA CGC TTA CGA GCC CG-3′; 2) 5′-TCA TTA TGA CGC TTA CGA GCC CG-3′; 3) 5′-TCA T(8-oxodG)A TGA CGC TTA CGA GCC CG-3′; 4) 5′-TCA (CPD)A TGA CGC TTA CGA GCC CG-3′ (CPD indicates cis-syn thymine dimer). These annealed fluorescent substrates were used in extension, single nucleotide incorporation, and steady-state kinetic assays. The following oligonucleotides were annealed at equal molar ratios for crystallization: 5′-AGC GTC AT-3′ and 5′-CAT GAT GAC GCT-3′; 5′-AGC GTC AT-3′ and 5′-CAT (8-oxodG)AT GAC GCT-3′.

      Extension, Single Nucleotide Incorporation, and Steady-state Kinetic Assays

      The extension assays were conducted with 5 μm DNA substrate (oligonucleotide), 1.2 μm hpol η, and 4 mm dNTP or rNTP mixtures (1 mm for each dNTP or rNTP) in 40 mm Tris-HCl buffer (pH 7.5) containing 10 mm dithiothreitol (DTT), 0.1 mg/ml bovine serum albumin (BSA), 5% glycerol (v/v), 5 mm MgCl2, and 100 mm KCl at 37 °C for 5, 30, 55, and 240 min. Single nucleotide incorporation assays were conducted using the same reaction buffer but with 5 μm annealed DNA substrate, 500 nm hpol η, and 1 mm each of dNTP or rNTP at 37 °C for 5, 30, and 55 min.
      For steady-state kinetic assays, 5 μm DNA substrate was incubated with 1.5 to 500 nm hpol η or the Y92A mutant as well as varying concentrations of each dNTP or rNTP at 37 °C for 5 min, in the same reaction buffer as extension and single base incorporation assay. Reactions were stopped by the addition of a quench buffer (90% formamide (v/v) and 10 mm EDTA). Products were separated on 18% (w/v) denaturing polyacrylamide gels and visualized using a Typhoon system (GE Healthcare). The data for steady-state kinetic assays were fit to a (hyperbolic) Michaelis-Menten equation using Prism software (GraphPad, La Jolla, CA).

      Crystallization, Diffraction, Data Collection, and Structure Determination

      Each DNA substrate and hpol η were mixed at a molar ratio of 1.1 to 1, before adding CaCl2 (final concentration 3.33 mm) and excess amount of rNTP. To obtain crystals by hanging drop vapor diffusion, 1 μl of each protein/DNA mixture was mixed with 1 μl of reservoir solution, which contained 100 mm sodium MES (pH 6.0), 5 mm CaCl2, and 16–22% (w/v) polyethylene glycol monomethyl ether 2000, followed by equilibration with 500 μl of the reservoir solution at 18 °C. The crystals were washed through cryoprotectant (composed of 3 volumes of reservoir solution and 1 volume of glycerol) before flash freezing in liquid nitrogen. Diffraction data were collected on the 21-ID-F or 21-ID-G beamline at the Advanced Photon Source (Life Sciences Collaborative Access Team, Argonne National Laboratory, Argonne, IL). Data were integrated and scaled using HKL2000 (
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      ) for molecular replacement phasing (PDB code 4O3N as the search model for hpol η·dG·rCTP, and PDB code 4O3P for hpol η·dG·rCTP and hpol η·dG·rATP) (
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      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
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      Results

      hpol η Inserts Ribonucleotides Opposite Undamaged Template dG or dA

      To assess the ribonucleotide incorporation ability of hpol η, we compared the extension of the primers in the presence of all four dNTPs or rNTPs with different incubation times. The primer was extended at 5 min and reached full-length after 30 min in the presence of rNTPs, compared with 5 min for full-length extension with dNTPs (Fig. 1A). To estimate the fidelity of ribonucleotide incorporation, hpol η was incubated with annealed undamaged DNA substrate as well as individual dNTP or rNTP. Under our reaction conditions, multiple nucleotides were added to the primer in the presence of a single dNTP in an error-prone manner. However, only one or two nucleotide extensions was observed with a single rNTP. Among the four ribonucleotides, rCTP (able to Watson-Crick pair with template dG) was the most efficient one for insertion (Fig. 1B).
      Figure thumbnail gr1
      FIGURE 1.hpol η incorporates dNTPs or rNTPs opposite an unmodified DNA template. A, full-length extension of the primer opposite unmodified DNA template (5 μm) with all four dNTPs or rNTPs by hpol η (1.2 μm) at 37 °C for 5, 30, 55, and 240 min (time gradients depicted with wedges). B, single nucleotide incorporation assays with 5 μm native primer-template DNA substrate, 500 nm hpol η, and 1 mm each of individual dNTP or rNTP at 37 °C for 5, 30, and 55 min.
      To further measure the efficiency and fidelity of rNTP incorporation by hpol η, steady-state kinetic experiments were conducted. The catalytic efficiency (kcat/Km) for rCTP insertion opposite template dG was 770-fold less than that for dCTP and 2–5-fold less relative to those for the mismatched dNTPs. However, rates of incorporation of the other ribonucleotides (other than rCTP) were very low and could not be measured experimentally. In addition, to investigate the effect of the template base, another set of DNA substrates with dT in the template instead of dG was included in the steady-state kinetic study. The change for catalytic efficiency between rATP and dATP insertion opposite template dT was 3400-fold, slightly higher than that between rCTP and dCTP opposite dG (770-fold, Table 1).
      TABLE 1Steady-state kinetics for insertion of nucleoside triphosphates opposite dG and dT by hpol η
      TemplatedNTP/rNTPKmkcatkcat/Kmf
      Misinsertion frequency is as follows: f = (kcat/Km)incorrect/(kcat/Km)correct.
      1/f
      Fold change is 1/f.
      μmmin−1μm−1 min−1
      dGdCTP2.4 ± 0.3119 ± 350 ± 611
      dATP84 ± 911 ± 10.13 ± 0.020.0026380
      dGTP58 ± 712 ± 10.21 ± 0.030.0042240
      dTTP170 ± 1658 ± 20.34 ± 0.030.0068150
      rCTP188 ± 1412 ± 10.064 ± 0.0070.0013770
      dTdCTP127 ± 819 ± 10.15 ± 0.010.0048210
      dATP2.5 ± 0.277 ± 231 ± 311
      dGTP9.1 ± 0.627 ± 13.0 ± 0.20.09710
      dTTP132 ± 1527 ± 10.20 ± 0.020.0065150
      rATP278 ± 372.5 ± 0.10.0090 ± 0.00120.000293400
      a Misinsertion frequency is as follows: f = (kcat/Km)incorrect/(kcat/Km)correct.
      b Fold change is 1/f.

      hpol η Inserts Ribonucleotides Opposite 8-oxodG

      We conducted primer extension assays by hpol η with 8-oxodG in the template strand in the presence of dNTPs or rNTPs. The pattern for primer extension past 8-oxodG was very similar to that against undamaged DNA (Figs. 1A and 2A). The primer was partially extended to full length by 30 min with the mixture of all four rNTPs, whereas hpol η elongated the primer to full length within 5 min with dNTPs. In single nucleotide insertion assays, only one rC was added to the primer within 5 min, although in the presence of the other individual rNTPs significantly elongated primers were only observed after 30 min of incubation (Fig. 2, A and B).
      Figure thumbnail gr2
      FIGURE 2.hpol η can bypass an 8-oxodG lesion and incorporate dNTPs or rNTPs. A, hpol η (1.2 μm) extended the primer against DNA template (5 μm) containing an 8-oxoG lesion in the presence of all four dNTPs or rNTPs at 37 °C for 5, 30, 55, and 240 min (time gradients depicted with wedges). B, single nucleotide incorporation assays with hpol η (500 nm) with 5 μm DNA substrate with 8-oxoG in the template and 1 mm each of dNTP or rNTP at 37 °C for 5, 30, and 55 min.
      In steady-state kinetic assays, hpol η incorporated dCTP opposite template 8-oxodG 2300-fold more efficiently than rCTP and 43,000-fold more than rATP. The difference between the catalytic efficiencies for rCTP and rATP incorporation was about 19, compared with 5 for dCTP and dATP, indicating that during nucleotide incorporation hpol η has similar base selectivity regardless of the sugar (Table 2).
      TABLE 2Steady-state kinetics for insertion of nucleoside triphosphates opposite 8-oxodG by hpol η
      TemplatedNTP/rNTPKmkcatkcat/Kmf1/f
      μmmin−1μm−1 min−1
      8-oxodGdCTP1.7 ± 0.381 ± 348 ± 911
      dATP8.4 ± 1.077 ± 39.2 ± 1.10.195.3
      dGTP18 ± 229 ± 11.6 ± 0.20.03330
      dTTP140 ± 1144 ± 10.31 ± 0.030.0065150
      rCTP292 ± 426.1 ± 0.30.021 ± 0.0030.000442300
      rATP445 ± 400.51 ± 0.020.0011 ± 0.00010.00002343000

      hpol η Incorporates Ribonucleotides Opposite CPD

      Given that hpol η is capable of bypassing CPD by inserting the correct nucleotide dA (
      • Masutani C.
      • Kusumoto R.
      • Yamada A.
      • Dohmae N.
      • Yokoi M.
      • Yuasa M.
      • Araki M.
      • Iwai S.
      • Takio K.
      • Hanaoka F.
      The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase η.
      ,
      • Biertümpfel C.
      • Zhao Y.
      • Kondo Y.
      • Ramón-Maiques S.
      • Gregory M.
      • Lee J.Y.
      • Masutani C.
      • Lehmann A.R.
      • Hanaoka F.
      • Yang W.
      Structure and mechanism of human DNA polymerase η.
      ), we investigated the incorporation of ribonucleotides against this lesion. hpol η extended the primer past CPD in a manner similar to that observed opposite undamaged DNA or 8-oxodG. With only rATP, more than 50% of the primers were elongated by two nucleotides after 5 min, much faster than with the other single rNTPs (Fig. 3, A and B). Quantitatively, rATP insertion was 1400-fold less efficient than dATP opposite CPD (Table 3).
      Figure thumbnail gr3
      FIGURE 3.hpol η can incorporate ribonucleotides opposite the CPD lesion and further extend the primer. A, extension of the primer opposite a DNA template (5 μm) containing a CPD lesion by hpol η (1.2 μm) in the presence of all four dNTPs or rNTPs at 37 °C for 5, 30, 55, and 240 min (time gradients depicted with wedges). B, extension of the primer by incubation of 5 μm DNA substrate with a CPD in the template strand, 500 nm hpol η, and 1 mm each of individual dNTP or rNTP at 37 °C for 5, 30, and 55 min.
      TABLE 3Steady-state kinetics for insertion of nucleoside triphosphates opposite CPD by hpol η
      TemplatedNTP/rNTPKmkcatkcat/Kmf1/f
      μmmin−1μm−1 min−1
      CPDdCTP31 ± 327 ± 10.87 ± 0.090.02638
      dATP1.7 ± 0.257 ± 134 ± 411
      dGTP34 ± 340 ± 11.2 ± 0.10.03529
      dTTP23 ± 232 ± 11.4 ± 0.10.04124
      rATP295 ± 357.0 ± 0.30.024 ± 0.0030.000711400

      hpol η Scaffolds the Incoming rCTP and Template dG to Form a Watson-Crick Base Pair with a Propeller Twist

      To understand the mechanism of ribonucleotide insertion by hpol η, we co-crystallized hpol η with an incoming rCTP positioned opposite template dG in the presence of Ca2+ (Table 4). The final Fourier (2FoFc) sum electron density map (with a threshold of 1σ) is shown in Fig. 4A. The incoming rCTP formed a Watson-Crick base pair with the template dG, but a significant propeller twist was observed. The dihedral angle between the two base planes was 27°. In addition, the base pair was slightly shifted toward the major groove, compared with the dG:dCTP pair at the hpol η active site. Phe-18 was identified as the steric gate residue, and the distance between either 2′-OH or 3′-OH of rCTP and their closest atoms of the Phe-18 side chain was 3.2 Å (Fig. 5). In comparison with the hpol η·dG:dCTP structure (PDB code 4O3N) (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ), the position of the phenyl ring of Phe-18 was almost identical. In addition, the distance between the 3′-OH of the primer end (two conformations) to Pα of the incoming rCTP was 4.5 or 4.3 Å, about 1 Å further than that in the hpol η·dG:dCTP structure (3.5 or 3.3 Å), providing an explanation for the 103-fold lower catalytic efficiency of the polymerization reaction (Fig. 5).
      TABLE 4Crystal data, data collection parameters, and structure refinement statistics
      Complexhpol η·dG:rCTPhpol η·(8-oxodG):rCTPhpol η·(8-oxodG):rATP
      Data collection
      Wavelength (Å)0.978560.978720.97856
      Space groupP61P61P61
      Resolution (Å)50.00-1.66 (1.69-1.66)
      Data shown in parentheses are from the highest resolution shell.
      50.00-1.78 (1.81-1.78)50.00-1.75 (1.78-1.75)
      Unit cell a = b, c (Å)99.28, 81.9099.12, 81.3899.02, 81.57
      Unique reflections54,114 (2700)43,104 (2117)45,786 (2302)
      Completeness (%)100.0 (100.0)99.2 (99.1)99.8 (100.0)
      I/σ(I)19.2 (2.4)18.5 (1.9)21.1 (2.2)
      Wilson B-factor (Å2)16.821.628.1
      R-merge
      R-merge is R linear = SUM(ABS(I − 〈I〉))/SUM (I).
      0.098 (0.978)0.115 (1.229)0.086 (0.909)
      Redundancy8.9 (8.8)8.8 (7.8)8.5 (7.3)
      Refinement
      R-work0.1714 (0.2757)0.1795 (0.2739)0.1781 (0.2766)
      R-free0.2074 (0.3021)0.2158 (0.3040)0.2150 (0.2778)
      No. of atoms
      Protein/DNA3376/4093304/4533303/390
      rNTP/Ca2+29/229/131/1
      H2O/glycerol444/12410/6374/0
      Protein residues427425425
      B-factor (Å)
      Average22.227.033.4
      Protein/DNA20.5/25.725.8/30.732.2/37.8
      rNTP/Ca2+16.3/17.319.5/15.629.7/23.6
      Water/glycerol31.7/24.833.4/20.439.1/-
      Root mean square deviations
      Bonds (Å)0.0070.0080.007
      Angles (°)1.0071.1461.023
      Ramachandran
      Favored (%)97.397.297.9
      Allowed (%)2.472.071.83
      Outliers (%)0.220.690.23
      PDB code5EWE5EWF5EWG
      a Data shown in parentheses are from the highest resolution shell.
      b R-merge is R linear = SUM(ABS(I − 〈I〉))/SUM (I).
      Figure thumbnail gr4
      FIGURE 4.Quality of the final Fourier (2FoFc) sum electron density drawn at the 1σ threshold around incoming ribonucleotides and the template base/lesions. A, hpol η·dG:rCTP. B, hpol η·(8-oxodG):rCTP. C, hpol η·dG:rATP.
      Figure thumbnail gr5
      FIGURE 5.Crystal structure of hpol η inserting rCTP opposite template dG in the presence of Ca2+. A, active site of the hpol η·dG:rCTP complex viewed from the major groove side. B, superimposition of the structures of the ternary hpol η·dG·rCTP and hpol η·dG·dCMPNPP (dCTP analog) (PDB code 4O3N) (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ) complexes, viewed from the major groove. C, dG:rCTP pair at the active site viewed from the top. D, top view of the superimposed active sites in the structures of hpol η·dG:rCTP and hpol η·dG:dCMPNPP complexes. For hpol η·dG:rCTP, the base pair dG:rCTP (as well as Ca2+) are highlighted in dark cyan, and the other nucleotides and key residues Arg-61, Gln-38, and Phe-18 are in light green. For hpol η·dG:dCTP, both the base pair G:dCMPNPP and Mg2+ are shown in orange, and the other nucleotides and Phe-18 are shown in khaki.

      hpol η Accommodates 8-oxodG in Two Conformations Opposite the Incoming rCTP

      We also crystallized hpol η with an incoming rCTP opposite 8-oxodG (Table 4). The final Fourier (2FoFc) sum electron density map is shown in Fig. 4B. Noticeably, the electron density of 8-oxodG did not fit either a pure anti conformation as seen at the active site of hpol η·(8-oxodG):dCTP or a pure syn conformation as in hpol η·(8-oxodG):dATP (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ). Instead, the electron density was indicative of two alternative conformations of 8-oxodG. After automated refinement using the program Phenix, the occupancy of 8-oxodG in the anti conformation refined to 50%, with the base in the syn conformation contributing the other half. In contrast to the dual conformation of 8-oxodG, the incoming rCTP fit the electron density very well, consistent with a single conformation (Fig. 4B). When 8-oxodG is in an anti conformation, the active site closely resembles that of the hpol η·dG:rCTP complex. Incoming rCTP and 8-oxodG are engaged in a Watson-Crick base pair with formation of a significant propeller twist. The dihedral angles of the bases of the incoming rCTP and 8-oxodG were 29° (anti conformation) and 25° (syn conformation) (Fig. 6). In the syn conformation, 8-oxodG maintained three H-bonds with rCTP as follows: O6 of 8-oxodG H-bonded with N4(H) of rCTP, with a distance of 2.8 Å, and N7(H) of 8-oxodG donated in a bifurcated H-bond to O2 and N3 of the incoming rCTP, with distances of 3.1 and 3.2 Å, respectively. The O8 atom of 8-oxodG in the syn conformation was positioned at 3.6 Å from O2 of rCTP and therefore outside the range for interacting.
      Figure thumbnail gr6
      FIGURE 6.Crystal structure of hpol η inserting rCTP opposite 8-oxodG. A, active site of the hpol η·(8-oxodG anti):rCTP complex viewed from the major groove side. B, superimposition of the structures of the ternary hpol η-(8-oxodG anti):rCTP and hpol η·(8-oxodG):dCMPNPP (PDB code 4O3P) (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ) complexes, viewed from the major groove. C, (8-oxodG anti):rCTP pair at the active site viewed from the top. D, top view of the superimposed active sites in the structures of the hpol η·(8-oxodG anti):rCTP and hpol η·(8-oxodG):dCMPNPP complexes. E, active site of the hpol η·(8-oxodG syn):rCTP complex viewed from the major groove side. F, superimposition of the structures of the ternary hpol η·(8-oxodG syn):rCTP and hpol η·(8-oxodG):dCMPNPP (PDB code 4O3P)(
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ) complexes, viewed from the major groove. G, (8-oxodG syn):rCTP pair at the active site viewed from the top. H, top view of the superimposed active sites in the same structures of the hpol η·(8-oxodG syn):rCTP and hpol η·(8-oxodG):dCMPNPP complexes. The color codes are the same as in and H-bonds are dashed lines.
      With either conformation adopted by 8-oxodG, the 8-oxodG:rCTP pair was shifted toward the major groove compared with the structure of hpol η·(8-oxodG):dCTP (PDB code 4O3P) (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ). The closest distance between 2′-OH of rCTP and Phe-18 was 3.3 Å, and the distance between 3′-OH and the steric gate residue was 3.4 Å. The 3′-OH of the primer end (two conformations) was 4.3 or 4.1 Å away from the Pα of rCTP, whereas the corresponding distance was 3.4 or 3.2 Å in the hpol η·(8-oxodG):dCTP complex, respectively, similar to the relative positions for rCTP and dCTP opposite template dG at the hpol η active site (Fig. 6).

      8-oxodG Adopts a syn Conformation in the Pair with the Incoming rATP at the hpol η Active Site

      Given that 8-oxodG adopts a syn-conformation in the structure of hpol η·(8-oxodG):dATP (PDB code 4O3O) (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ), we also examined the orientation of rATP opposite 8-oxodG at the active site of hpol η. The final Fourier (2FoFc) sum electron density map around the base pair at the active site is shown in Fig. 4C. 8-oxodG indeed adopted a syn conformation, but instead of a planar Hoogsteen base pair, a propeller twist of 32° was observed for 8-oxodG:rATP. As a result, the distance between O6 of the lesion and N6 of rATP was 3.7 Å, too long for formation of an H-bond. Because of the propeller twist, 2′-OH of rATP was 3.1 Å away from the closet atom of Phe-18, whereas the closest distance between 3′-OH and the side chain of the steric gate residue was 3.2 Å. In the hpol η·(8-oxodG):rATP complex, 8-oxodG moved closer to the backbone of the template strand, whereas rATP shifted further into the major groove, compared with the hpol η·(8-oxodG):dATP complex. The distance between 3′-OH of the primer end to Pα of rATP was 4.4 Å. By comparison, the corresponding distance in the hpol η·(8-oxodG):dATP complex was 3.3 or 3.1 Å (Fig. 7).
      Figure thumbnail gr7
      FIGURE 7.Crystal structure of hpol η inserting rATP opposite 8-oxodG. A, active site of the hpol η·(8-oxodG):rATP complex viewed from the major groove side. B, superimposition of the structures of the ternary hpol η·(8-oxodG):rATP and hpol η·(8-oxodG):dAMPNPP (PDB code 4O3O) (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ) complexes. C, (8-oxodG):rATP pair at the active site viewed from the top. D, top view of the superimposed active sites in the structures of the hpol η·(8-oxodG):rATP and hpol η·(8-oxodG):dAMPNPP complexes. The color codes are the same as in .

      Tyr-92 Acts as a Second Line of Defense to Stabilize the Steric Gate Residue Phe-18 at the Active Site of hpol η

      Superimpositions of the crystal structure with incoming rNTP and the corresponding structure with incoming dNTP revealed that the side chain of Phe-18 adopts an almost identical position in both cases. Upon closer investigation, a second guard residue, Tyr-92, was identified. The side chain of Tyr-92 is capable of forming a π-π interaction with the phenyl ring of Phe-18, with a distance of about 3.9 Å (Fig. 8). This stacking interaction appears to stabilize the side chain of Phe-18, leaving it in position and preventing the incoming nucleotide from sliding into the minor groove.
      Figure thumbnail gr8
      FIGURE 8.Second line of defense in hpolη: Tyr-92 stabilizes the steric gate residue Phe-18 by π-π interaction. A, steric gate residue Phe-18 and the second line of defense residue Tyr-92 in the hpol η·dG:rCTP complex viewed from the side. B, view of Phe-18 and Tyr-92 in the superimposition of the structures of hpol η·dG:rCTP and hpol η·dG:dCMPNPP (PDB code 4O3N) (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ). In hpol η·dG:rCTP, Phe-18 and Tyr-92 are shown in green, and rCTP and Ca2+ are in dark cyan. In hpol η·dG:dCMPNPP, Phe-18 and Tyr-92 are shown in khaki, and dCMPNPP and Mg2+ in shown in orange.
      To address this hypothesis, we used site-directed mutagenesis to introduce the mutation Y92A in hpol η and examined its activity in steady-state kinetic assays opposite template dG. The catalytic efficiency (kcat/Km) of the mutant Y92A for dCTP insertion (1.6 μm−1 min−1) was 67-fold higher than for rCTP (0.024 μm−1 min−1), in comparison with the 770-fold difference for wild-type hpol η (Table 1), indicating that the introduction of the Y92A mutation reduced the sugar discrimination ability of hpol η.

      Discussion

      Cellular rNTP concentrations are much higher than dNTP concentrations, thus presenting DNA polymerases with a challenge to discriminate against the former (
      • Nick McElhinny S.A.
      • Kumar D.
      • Clark A.B.
      • Watt D.L.
      • Watts B.E.
      • Lundström E.B.
      • Johansson E.
      • Chabes A.
      • Kunkel T.A.
      Genome instability due to ribonucleotide incorporation into DNA.
      • Traut T.W.
      Physiological concentrations of purines and pyrimidines.
      ,
      • Nick McElhinny S.A.
      • Watts B.E.
      • Kumar D.
      • Watt D.L.
      • Lundström E.B.
      • Burgers P.M.
      • Johansson E.
      • Chabes A.
      • Kunkel T.A.
      Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.
      • Chabes A.
      • Stillman B.
      Constitutively high dNTP concentration inhibits cell cycle progression and the DNA damage checkpoint in yeast Saccharomyces cerevisiae.
      ). How DNA polymerases regulate rNTP incorporation has been studied for at least 20 years (
      • Nick McElhinny S.A.
      • Kumar D.
      • Clark A.B.
      • Watt D.L.
      • Watts B.E.
      • Lundström E.B.
      • Johansson E.
      • Chabes A.
      • Kunkel T.A.
      Genome instability due to ribonucleotide incorporation into DNA.
      ,
      • Nick McElhinny S.A.
      • Watts B.E.
      • Kumar D.
      • Watt D.L.
      • Lundström E.B.
      • Burgers P.M.
      • Johansson E.
      • Chabes A.
      • Kunkel T.A.
      Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.
      ,
      • Kirouac K.N.
      • Suo Z.
      • Ling H.
      Structural mechanism of ribonucleotide discrimination by a Y-family DNA polymerase.
      • Donigan K.A.
      • McLenigan M.P.
      • Yang W.
      • Goodman M.F.
      • Woodgate R.
      The steric gate of DNA polymerase ι regulates ribonucleotide incorporation and deoxyribonucleotide fidelity.
      ,
      • Gosavi R.A.
      • Moon A.F.
      • Kunkel T.A.
      • Pedersen L.C.
      • Bebenek K.
      The catalytic cycle for ribonucleotide incorporation by human DNA pol λ.
      ,
      • Clausen A.R.
      • Murray M.S.
      • Passer A.R.
      • Pedersen L.C.
      • Kunkel T.A.
      Structure-function analysis of ribonucleotide bypass by B family DNA replicases.
      ,
      • Clausen A.R.
      • Zhang S.
      • Burgers P.M.
      • Lee M.Y.
      • Kunkel T.A.
      Ribonucleotide incorporation, proofreading and bypass by human DNA polymerase δ.
      • Cavanaugh N.A.
      • Beard W.A.
      • Wilson S.H.
      DNA polymerase β ribonucleotide discrimination: insertion, misinsertion, extension, and coding.
      ). In general, a steric gate residue has been considered to constitute a physical barrier to prevent ribonucleotide incorporation by individual DNA polymerases (
      • Joyce C.M.
      Choosing the right sugar: how polymerases select a nucleotide substrate.
      • DeLucia A.M.
      • Chaudhuri S.
      • Potapova O.
      • Grindley N.D.
      • Joyce C.M.
      The properties of steric gate mutants reveal different constraints within the active sites of Y-family and A-family DNA polymerases.
      ,
      • Vaisman A.
      • Kuban W.
      • McDonald J.P.
      • Karata K.
      • Yang W.
      • Goodman M.F.
      • Woodgate R.
      Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity.
      ,
      • DeLucia A.M.
      • Grindley N.D.
      • Joyce C.M.
      An error-prone family Y DNA polymerase (DinB homolog from Sulfolobus solfataricus) uses a “steric gate” residue for discrimination against ribonucleotides.
      ,
      • Niimi N.
      • Sassa A.
      • Katafuchi A.
      • Grúz P.
      • Fujimoto H.
      • Bonala R.R.
      • Johnson F.
      • Ohta T.
      • Nohmi T.
      The steric gate amino acid tyrosine 112 is required for efficient mismatched-primer extension by human DNA polymerase κ.
      ,
      • Yang G.
      • Franklin M.
      • Li J.
      • Lin T.C.
      • Konigsberg W.
      A conserved Tyr residue is required for sugar selectivity in a pol α DNA polymerase.
      ,
      • Kirouac K.N.
      • Suo Z.
      • Ling H.
      Structural mechanism of ribonucleotide discrimination by a Y-family DNA polymerase.
      • Donigan K.A.
      • McLenigan M.P.
      • Yang W.
      • Goodman M.F.
      • Woodgate R.
      The steric gate of DNA polymerase ι regulates ribonucleotide incorporation and deoxyribonucleotide fidelity.
      ). The results of site-directed mutagenesis experiments illustrate the importance of the steric gate effect. For example, the Y39A mutation in hpol ι causes the enzyme to almost totally lose its ability to discriminate between the ribose and deoxyribose sugars (
      • Donigan K.A.
      • McLenigan M.P.
      • Yang W.
      • Goodman M.F.
      • Woodgate R.
      The steric gate of DNA polymerase ι regulates ribonucleotide incorporation and deoxyribonucleotide fidelity.
      ). Also, the Sulfolobus solfataricus DNA polymerase Dpo4 Y12A mutant is capable of incorporating ribonucleotides into primers, and alanine, in place of the steric gate residue Tyr-12, allows space for the 2′-OH of the incoming ribonucleotide, as seen in the x-ray crystal structure (
      • Kirouac K.N.
      • Suo Z.
      • Ling H.
      Structural mechanism of ribonucleotide discrimination by a Y-family DNA polymerase.
      ). A recently published study of yeast pol η shows that mutation of the steric gate residue Phe-35 to an alanine leads to increased ability of ribonucleotide incorporation (
      • Donigan K.A.
      • Cerritelli S.M.
      • McDonald J.P.
      • Vaisman A.
      • Crouch R.J.
      • Woodgate R.
      Unlocking the steric gate of DNA polymerase η leads to increased genomic instability in Saccharomyces cerevisiae.
      ).

      2′-OH of the Incoming Ribonucleotide Leads to a Propeller Twist between the Paired Bases at the Active Site of hpol η and Slows the Reaction

      We crystallized wild-type hpol η with primer-template DNA duplexes containing either dG or 8-oxodG opposite the incoming rNTP. Interestingly, the extra 2′-OH of the incoming rNTP does not directly point into the phenyl ring of the steric gate residue. Instead, to avoid close contact, in the structure of rCTP opposite template dG, rCTP undergoes a shift relative to the position of dCTP in the corresponding crystal structure. However, the sugar pucker of the incoming nucleotide is of the C3′-endo type in both cases. Because of the shift of rCTP, a significant propeller twist occurs between cytosine of the incoming nucleotide and guanine of the template (Fig. 5). Similar situations were observed in the crystal structures of the hpol η·(8-oxodG):rCTP and hpol η·(8-oxodG):rATP complexes (FIGURE 6., FIGURE 7.). The dihedral angle between the planes of the two bases in the three structures varies between 25 and 32°, compared with 3° in the hpol η·G:dCTP complex (PDB code 403N) (
      • Patra A.
      • Nagy L.D.
      • Zhang Q.
      • Su Y.
      • Müller L.
      • Guengerich F.P.
      • Egli M.
      Kinetics, structure, and mechanism of 8-oxo-7,8-dihydro-2′-deoxyguanosine bypass by human DNA polymerase η.
      ).
      These particular structures accommodated by hpol η are a consequence of its unique, unusually spacious active site that is relatively open on the major groove side. For example, a similar shift by the ribose sugar and the resulting significant propeller twist of the base pair at the active site were not observed in the structure of the wild-type hpol β·DNA:rCTP complex (PDB code 3RH4) (
      • Cavanaugh N.A.
      • Beard W.A.
      • Batra V.K.
      • Perera L.
      • Pedersen L.G.
      • Wilson S.H.
      Molecular insights into DNA polymerase deterrents for ribonucleotide insertion.
      ). Noticeably, in each of the three structures in our study, the distance between the 3′-OH of the primer end to the Pα of the incoming ribonucleotide is increased by 1 Å compared with the corresponding complexes with incoming dNTPs. As a result, the energy barrier for the nucleotidyl transfer reaction is elevated, and the overall reaction rate is reduced by about 3 orders of magnitude.

      hpol η Maintains Base Discrimination When the Incoming Nucleotide Is a Ribonucleotide

      Our results show that hpol η is still capable of discriminating bases among incoming rNTPs. The Watson-Crick base pair between an incoming rCTP and template dG is maintained, albeit with a propeller twist between bases because of different positioning of sugars of rNTPs relative to dNTPs (Fig. 5). Even with a DNA lesion (8-oxodG or CPD) on the template strand, hpol η still displays relatively high selectivity for the incoming nucleotide as a result of a generally conserved base pairing preference. Thus, it is noteworthy that the catalytic efficiency for rCTP incorporation opposite 8-oxodG is 19-fold higher than that of rATP insertion. By comparison, the change between the catalytic efficiencies of the correct insertion of dCTP opposite the lesion and erroneous insertion of dATP is only 5-fold. In addition, the orientations of 8-oxodG in the template strand vary somewhat in the crystal structures of the hpol η·(8-oxoG):rCTP and hpol η·(8-oxoG)·rATP ternary complexes (FIGURE 6., FIGURE 7.). These results indicate that the incoming rNTP may influence the position and conformation of the template lesion to some extent and therefore affect base selectivity.

      Second Line of Defense That Stabilizes the Steric Gate Residue Has Been Observed in Other DNA Polymerases

      In our study, we identified Tyr-92 as the second “guard” stabilizing the steric gate residue Phe-18 by π-π interaction, i.e. loss of Tyr-92 makes the polymerase an order of magnitude less discriminating. This second line of defense is also seen in some other DNA polymerases, in comparing their structures. For example, hpol ι Tyr-102 acts as the second guard, stacking with the steric gate residue Tyr-39 (
      • Donigan K.A.
      • McLenigan M.P.
      • Yang W.
      • Goodman M.F.
      • Woodgate R.
      The steric gate of DNA polymerase ι regulates ribonucleotide incorporation and deoxyribonucleotide fidelity.
      ). In hpol κ, Tyr-112 constitutes the steric gate by forming a stacking interaction with Tyr-174 (
      • Vasquez-Del Carpio R.
      • Silverstein T.D.
      • Lone S.
      • Swan M.K.
      • Choudhury J.R.
      • Johnson R.E.
      • Prakash S.
      • Prakash L.
      • Aggarwal A.K.
      Structure of human DNA polymerase kappa inserting dATP opposite an 8-oxoG DNA lesion.
      ). Similarly, in DNA polymerase Dpo4, a pol κ homolog from S. solfataricus, the steric gate residue Tyr-12 is accompanied by Tyr-81 that serves as the second line of defense (
      • Vaisman A.
      • Ling H.
      • Woodgate R.
      • Yang W.
      Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis.
      ). Conversely, the replicative B-family bacteriophage RB69 uses a single DNA polymerase steric gate residue, Tyr-416, packed against an α-helix (
      • Clausen A.R.
      • Murray M.S.
      • Passer A.R.
      • Pedersen L.C.
      • Kunkel T.A.
      Structure-function analysis of ribonucleotide bypass by B family DNA replicases.
      ).

      Biological Relevance of Ribonucleotide Incorporation by hpol η

      Considering that the cellular concentration of rNTPs is 101–106-fold higher that that of dNTPs (
      • Nick McElhinny S.A.
      • Kumar D.
      • Clark A.B.
      • Watt D.L.
      • Watts B.E.
      • Lundström E.B.
      • Johansson E.
      • Chabes A.
      • Kunkel T.A.
      Genome instability due to ribonucleotide incorporation into DNA.
      • Traut T.W.
      Physiological concentrations of purines and pyrimidines.
      ,
      • Nick McElhinny S.A.
      • Watts B.E.
      • Kumar D.
      • Watt D.L.
      • Lundström E.B.
      • Burgers P.M.
      • Johansson E.
      • Chabes A.
      • Kunkel T.A.
      Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases.
      • Chabes A.
      • Stillman B.
      Constitutively high dNTP concentration inhibits cell cycle progression and the DNA damage checkpoint in yeast Saccharomyces cerevisiae.
      ) and the catalytic efficiencies for ribonucleotide insertion are only about 103-fold lower than those for deoxynucleotides, it is highly possible that hpol η inserts a considerable amount of ribonucleotides into DNA as part of the various biological processes in which it is involved.
      As a member of the DNA polymerase Y-family, one of the most important roles of hpol η is the bypass of lesions during DNA replication. The base selectivity of hpol η mainly depends on the type of the template base/lesion and the flanking DNA sequence (e.g. results in Tables 13). However, in this study, we also investigated the ability of hpol η to discriminate between ribose and deoxyribose sugars of the incoming nucleotides, especially opposite the DNA lesions 8-oxodG and CPD in the template strand. Judging from our study, the active site of hpol η can simultaneously accommodate a lesion on the template strand and a ribo-sugar of the incoming nucleotide, while still maintaining significant base selectivity.

      Author Contributions

      Y. S. designed and conducted the experiments, crystallized the proteins, and solved the structures. Y. S., F. P. G., and M. E. conceived the studies, analyzed the results, and wrote the paper.

      Acknowledgments

      We thank Drs. J. M. Harp, P. S. Pallan, and E. A. Kowal for advice on the x-ray crystallography work. We also thank K. Trisler for assistance in the preparation of the manuscript. Vanderbilt University is a member institution of the Life Sciences Collaborative Access Team at sector 21 of the Advanced Photon Source, Argonne, IL. Use of the Advanced Photon Source at Argonne National Laboratory was supported by the United States Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-06CH11357.

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