Lesion Bypass of N2-Ethylguanine by Human DNA Polymerase ι
2008; Elsevier BV; Volume: 284; Issue: 3 Linguagem: Inglês
10.1074/jbc.m807296200
ISSN1083-351X
AutoresMatthew G. Pence, Patrick Blans, Charles N. Zink, Thomas Hollis, James C. Fishbein, Fred W. Perrino,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoNucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase ι was measured and structures of the DNA polymerase ι-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase ι opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase ι incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase ι opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase ι extends from the N2-ethyl-Gua:Cyt 3′ terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase ι catalyzed reaction is well suited for N2-ethyl-Gua bypass. The structure of DNA polymerase ι with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase ι complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase ι polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase ι efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration. Nucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase ι was measured and structures of the DNA polymerase ι-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase ι opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase ι incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase ι opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase ι extends from the N2-ethyl-Gua:Cyt 3′ terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase ι catalyzed reaction is well suited for N2-ethyl-Gua bypass. The structure of DNA polymerase ι with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase ι complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase ι polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase ι efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration. N2-Ethylguanine (N2-ethyl-Gua) 2The abbreviations used are: N2-ethyl-Gua, N2-ethylguanine; DNA pol ι, DNA polymerase ι; PAD, polymerase-associated domain; pol, polymerase; MES, 4-morpholineethanesulfonic acid.2The abbreviations used are: N2-ethyl-Gua, N2-ethylguanine; DNA pol ι, DNA polymerase ι; PAD, polymerase-associated domain; pol, polymerase; MES, 4-morpholineethanesulfonic acid. is an acetaldehyde-derived DNA adduct generated from the reduction of acetaldehyde with 2′-deoxyguanosine-3′-monophosphate (1Fang J.L. Vaca C.E. Carcinogenesis. 1995; 16: 2177-2185Crossref PubMed Scopus (118) Google Scholar). Humans are exposed to acetaldehyde from the environment and through the formation of acetaldehyde by the oxidation of ethanol (2Fang J.L. Vaca C.E. Carcinogenesis. 1997; 18: 627-632Crossref PubMed Scopus (243) Google Scholar). N2-Ethyl-Gua has been detected in the DNA of both alcoholic and nonalcohol drinkers (2Fang J.L. Vaca C.E. Carcinogenesis. 1997; 18: 627-632Crossref PubMed Scopus (243) Google Scholar, 3Matsuda T. Terashima I. Matsumoto Y. Yabushita H. Matsui S. Shibutani S. Biochemistry. 1999; 38: 929-935Crossref PubMed Scopus (81) Google Scholar). Ethanol is classified as a human carcinogen, and acetaldehyde is known to contribute to the formation of malignant tumors (4Baan R. Straif K. Grosse Y. Secretan B. El Ghissassi F. Bavard V. Altieri A. Cogliano V. Lancet Oncol. 2007; 8: 292-293Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar). The formation of N2-ethyl-Gua during the reduction of acetaldehyde could cause ethanol-related cancers (5Seitz H.K. Becker P. Alcohol Res. Health. 2007; 30: 38-47PubMed Google Scholar). The ethyl moiety of N2-ethyl-Gua is predicted to project into the minor groove of duplex DNA. The N2-ethyl-Gua adduct is a strong block to DNA replication by replicative DNA polymerases in vitro and in cells (6Perrino F.W. Blans P. Harvey S. Gelhaus S.L. McGrath C. Akman S.A. Jenkins G.S. Lacourse W.R. Fishbein J.C. Chem. Res. Toxicol. 2003; 16: 1616-1623Crossref PubMed Scopus (44) Google Scholar, 7Upton D.C. Wang X. Blans P. Perrino F.W. Fishbein J.C. Akman S.A. Chem. Res. Toxicol. 2006; 19: 960-967Crossref PubMed Scopus (28) Google Scholar). Structures of bacteriophage DNA polymerase (pol) RB69, a homolog of human DNA pol α, indicate a possible mechanism of N2-ethyl-Gua blocked DNA replication. The structures reveal a DNA-binding motif that contacts the DNA minor groove and functions as an important safeguard to replication fidelity (8Franklin M.C. Wang J. Steitz T.A. Cell. 2001; 105: 657-667Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). The blocking of replicative DNA pols by N2-ethyl-Gua could arise when the ethyl group, protruding into the minor groove, disrupts protein:DNA contacts involved in the proposed "checking mechanism" (8Franklin M.C. Wang J. Steitz T.A. Cell. 2001; 105: 657-667Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). N2-Ethyl-Gua also has a high mis-coding potential during DNA replication with the Klenow fragment of Escherichia coli DNA pol I (9Terashima I. Matsuda T. Fang T.W. Suzuki N. Kobayashi J. Kohda K. Shibutani S. Biochemistry. 2001; 40: 4106-4114Crossref PubMed Scopus (46) Google Scholar). Mutations caused by N2-ethyl-Gua range from single base deletions to transversions (10Upton D.C. Wang X. Blans P. Perrino F.W. Fishbein J.C. Akman S.A. Mutat. Res. 2006; 599: 1-10Crossref PubMed Scopus (13) Google Scholar). The Y family DNA polymerases η, ι, and κ replicate through adducted DNA templates (6Perrino F.W. Blans P. Harvey S. Gelhaus S.L. McGrath C. Akman S.A. Jenkins G.S. Lacourse W.R. Fishbein J.C. Chem. Res. Toxicol. 2003; 16: 1616-1623Crossref PubMed Scopus (44) Google Scholar, 11Choi J.Y. Guengerich F.P. J. Biol. Chem. 2006; 281: 12315-12324Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 12Choi J.Y. Guengerich F.P. J. Mol. Biol. 2005; 352: 72-90Crossref PubMed Scopus (79) Google Scholar, 13Choi J.Y. Angel K.C. Guengerich F.P. J. Biol. Chem. 2006; 281: 21062-21072Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and an open, more rigid active site contributes to lesion bypass (14Prakash S. Johnson R.E. Prakash L. Annu. Rev. Biochem. 2005; 74: 317-353Crossref PubMed Scopus (813) Google Scholar). The multitude of Y family DNA pols suggests that a variety of mechanisms might be utilized by these polymerases during lesion bypass dependent upon the nature of the specific DNA adducts. Structural data indicate that DNA pol ι rotates unadducted template purines from the anti to syn conformation in ternary complexes and forms hydrogen bonds between the Hoogsteen edge of the template base and the Watson-Crick edge of the incoming nucleotide (15Nair D.T. Johnson R.E. Prakash S. Prakash L. Aggarwal A.K. Nature. 2004; 430: 377-380Crossref PubMed Scopus (253) Google Scholar, 16Nair D.T. Johnson R.E. Prakash L. Prakash S. Aggarwal A.K. Structure. 2005; 13: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 17Nair D.T. Johnson R.E. Prakash L. Prakash S. Aggarwal A.K. Structure. 2006; 14: 749-755Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Kinetic studies show that DNA pol ι has increased efficiency and fidelity during nucleotide insertion opposite template purines (11Choi J.Y. Guengerich F.P. J. Biol. Chem. 2006; 281: 12315-12324Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 18Johnson R.E. Washington M.T. Haracska L. Prakash S. Prakash L. Nature. 2000; 406: 1015-1019Crossref PubMed Scopus (573) Google Scholar, 19Johnson R.E. Haracska L. Prakash L. Prakash S. Mol. Cell. Biol. 2006; 26: 6435-6441Crossref PubMed Scopus (30) Google Scholar, 20Perrino F.W. Harvey S. Blans P. Gelhaus S. Lacourse W.R. Fishbein J.C. Chem. Res. Toxicol. 2005; 18: 1451-1461Crossref PubMed Scopus (11) Google Scholar). Similarly, rotation of the template base to the syn conformation is observed in the structure of DNA pol ι complexed with the 1,N6-ethenodeoxyadenosine lesion, allowing correct nucleotide insertion but not subsequent extension opposite the adduct (21Nair D.T. Johnson R.E. Prakash L. Prakash S. Aggarwal A.K. Nat. Struct. Mol. Biol. 2006; 13: 619-625Crossref PubMed Scopus (96) Google Scholar). Rotation of the purine base at the active site of DNA pol ι would allow for efficient bypass of DNA adducts at the N2 of Gua by repositioning the adduct into the major groove and removing potential steric overlap between the lesion and incoming nucleotide. Thus, DNA pol ι could be involved in the bypass of the minor groove DNA adduct N2-ethyl-Gua. The DNA polymerases utilize two divalent metal ions for activation of catalysis (22Beese L.S. Friedman J.M. Steitz T.A. Biochemistry. 1993; 32: 14095-14101Crossref PubMed Scopus (169) Google Scholar, 23Beese L.S. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (909) Google Scholar, 24Yang W. Lee J.Y. Nowotny M. Mol. Cell. 2006; 22: 5-13Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). The metals play a role in binding and positioning of the incoming nucleotide and in determining fidelity during catalysis (24Yang W. Lee J.Y. Nowotny M. Mol. Cell. 2006; 22: 5-13Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 25Garcia-Diaz M. Bebenek K. Krahn J.M. Pedersen L.C. Kunkel T.A. DNA Repair (Amst.). 2007; 6: 1333-1340Crossref PubMed Scopus (55) Google Scholar). The Mg2+ ion is often used as the activating metal for DNA polymerization studies in vitro (24Yang W. Lee J.Y. Nowotny M. Mol. Cell. 2006; 22: 5-13Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). The Mn2+ ion also binds to and activates DNA polymerases but frequently results in decreased fidelity of the replicative DNA polymerases (26Sirover M.A. Loeb L.A. Science. 1976; 194: 1434-1436Crossref PubMed Scopus (364) Google Scholar, 27Sirover M.A. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2331-2335Crossref PubMed Scopus (79) Google Scholar). Recently, the Mn2+ ion has been shown to increase the efficiency and fidelity of nucleotide incorporation by DNA pol ι opposite a template Thy nucleotide (28Frank E.G. Woodgate R. J. Biol. Chem. 2007; 282: 24689-24696Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The strong blocking effect of N2-ethyl-Gua to the replicative DNA polymerases and its possible role in alcohol-related cancers have prompted our studies on bypass of N2-ethyl-Gua by the Y family DNA pol ι. These data provide new insights into replication bypass of the ethanol-derived N2-ethyl-Gua adduct with potential carcinogenic consequences. The structures of DNA pol ι complexed with N2-ethyl-Gua containing DNA provide direct evidence for the initial anti position of the N2-ethyl-Gua that is subsequently rotated into the syn position upon binding the correct Cyt nucleotide but not upon binding the incorrect Thy nucleotide. The N2-ethyl moiety is easily accommodated in the major groove binding pocket of DNA pol ι by the specific repositioning of Lys309 located in a loop of the PAD domain. The Lys309 hydrogen bonding to the 5′ phosphate of the N2-ethyl-Gua template base in the anti orientation repositions to accommodate the ethyl side chain. This repositioning of Lys309 defines the available space for accommodation of relatively small adducts such as the alkyl lesions at the N2 position of Gua for efficient replication past these lesions by DNA pol ι. Furthermore, we show that when Mn2+ is the activating divalent metal, DNA pol ι bypass of N2-ethyl-Gua occurs with increased efficiency but reduced fidelity compared with Mg2+, demonstrating that Mn2+ could play an important role in modulating efficiency and fidelity of lesion bypass of minor groove purine adducts like N2-ethyl-Gua by the Y family DNA polymerases. Consequences of Mn2+ as the activating metal and flexibility of the DNA pol ι PAD domain for efficient bypass of N2-ethyl-Gua are discussed. Oligonucleotides—N2-Ethyl-Gua phosphoramidites and template oligonucleotides were prepared as described previously (6Perrino F.W. Blans P. Harvey S. Gelhaus S.L. McGrath C. Akman S.A. Jenkins G.S. Lacourse W.R. Fishbein J.C. Chem. Res. Toxicol. 2003; 16: 1616-1623Crossref PubMed Scopus (44) Google Scholar). Three DNA primer oligonucleotides, 5′-(6-FAM)-GCTCCGGAACCC-3′, 5′-(6-FAM)-GCTCCGGAACCCTT-3′, and 5′-(6-FAM)-GCTCCGGAACCCTTC-3′, were purchased from Operon Biotechnologies, Inc. (Huntsville, AL). For crystallization experiments, a self-annealing DNA oligonucleotide containing the N2-ethyl-Gua adduct and a dideoxy CMP at the 3′ end, 5′-TCTXGGGTCCTAGGACCddC-3′ (where X = N2-ethyl-Gua), was synthesized by Midland Certified Reagents (Midland, TX) using the supplied N2-ethyl-Gua phosphoramidites. Synthesis was carried out using cyanoethyl phosphoramidite chemistry, and protecting groups were removed by hydrolysis with concentrated ammonium hydroxide (20Perrino F.W. Harvey S. Blans P. Gelhaus S. Lacourse W.R. Fishbein J.C. Chem. Res. Toxicol. 2005; 18: 1451-1461Crossref PubMed Scopus (11) Google Scholar). The oligo was purified by reverse phase high performance liquid chromatography (mass calculated = 5503.7, mass observed = 5504.9). Expression and Purification of Human DNA Polymerase ι— The recombinant catalytic fragment of human DNA pol ι (amino acids 1–420) was made as an maltose-binding protein-DNA pol ι fusion protein with a PreScission Protease cleavage site seven residues from the DNA pol ι N-terminal methionine. The PreScission Protease recognition sequence and DNA pol ι coding sequence were verified by DNA sequencing. The plasmid constructs were transformed into E. coli BL21(DE3) Rosetta 2 cells (Novagen) for overexpression. Cells were grown to an A600 = 0.5 at 37 °C and quickly cooled on ice to 17 °C. After induction with 1 mm isopropyl β-d-thiogalactopyranoside, the cells were allowed to grow for 15 h at 17 °C. Cell extracts were prepared and the maltose-binding protein-DNA pol ι fusion protein was bound to an amylose resin in buffer containing 20 mm Tris-HCl (pH 7.5), 1 mm EDTA, and 200 mm NaCl. The fusion protein was cleaved overnight by on-column incubation with PreScission Protease at 4 °C. The recovered DNA pol ι was purified to homogeneity using phosphocellulose chromatography. Assays—For primer extension assays the DNA primer (12-mer) was hybridized to the 32-mer DNA template and added to reactions containing 20 mm Tris-HCl (pH 7.5), 2 mm dithiothreitol, 100 μm dNTP, 10 nm DNA pol ι, and the amount of MgCl2 or MnCl2 indicated in the figure legends. Incubations were for 15 min at 37 °C and reactions were quenched with EtOH. Samples were dried and resuspended in 5 μl of a 95% formamide/dye solution. Extension products were separated on 23% urea-polyacrylamide gels, and imaged with a PhosphorImager (Molecular Dynamics) and quantified using ImageQuant software. For the kinetic assays, the site-specific insertion procedure of Boosalis et al. (29Boosalis M.S. Petruska J. Goodman M.F. J. Biol. Chem. 1987; 262: 14689-14696Abstract Full Text PDF PubMed Google Scholar) was used. DNA primers (14-mer for insertion and 15-mer for extension) were hybridized to the 32-mer DNA templates and added to reactions containing 20 mm Tris-HCl (pH 7.5), 2 mm dithiothreitol, 2 mm MgCl2 or 0.075 mm MnCl2, 50 nm primer-template, and 0.625 nm DNA pol ι (Mg2+-activated reactions), or 0.2 nm DNA pol ι (Mn2+-activated reactions). The amounts of DNA pol ι in reactions yielded ∼20% extended product maximally. Incubations were for 10 min at 37 °C and reactions were processed as described above. All extended product bands were used to determine kinetic parameters (Km and kcat values) by non-linear regression using SigmaPlot 8.02 software (SPSS Science, Inc.). Relative insertion frequencies were calculated as 1/[(kcat/KM,correct)/(kcat/KM,incorrect)]. Crystallization of Human DNA Pol ι—The purified catalytic fragment of DNA pol ι was dialyzed into 20 mm NaPO4 monobasic, 1 mm EDTA, 1 mm dithiothreitol, and 150 mm NaCl and concentrated to ∼11 mg/ml. The DNA pol ι was mixed at a 1:1.2 molar ratio with the N2-ethyl-Gua containing DNA oligonucleotide. To study ternary complexes, MgCl2 and dCTP (or dTTP) were added to final concentrations of 10 and 20 mm, respectively. Crystals grew from solutions described by Nair et al. (16Nair D.T. Johnson R.E. Prakash L. Prakash S. Aggarwal A.K. Structure. 2005; 13: 1569-1577Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) containing 0.2–0.4 m (NH4)2SO4, 12.5–15% PEG 5000 monomethyl ether, and 0.1 m MES (pH 6.5). Crystal trays were kept at 4 °C and diffraction quality crystals appeared in 1–3 days. The crystals belonged to space group P6522 and had cell dimensions of a = b = 98.53 Å, c = 202.35 Å for dCTP containing crystals, and a = b = 98.64 Å, c = 202.23 Å for dTTP containing crystals, and α = β = 90°, γ = 120°. For data collection crystals were step soaked for 5 min in mother liquor solutions containing 0–25% glycerol and flash frozen in liquid nitrogen. Structure Determination and Refinement—X-ray diffraction data were collected using CuKα radiation from an in-house MicroMax 007 generator on a Saturn 92 CCD detector (Rigaku). The data were indexed, integrated, and scaled using d*TREX (30Pflugrath J.W. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1409) Google Scholar), and phases were calculated using molecular replacement. Molecular replacement with Phaser (31McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1590) Google Scholar) generated a unique solution using DNA pol ι (Protein Data Bank code 2ALZ) minus DNA as a search model. Electron density maps calculated to 2.5 Å (dCTP) and 2.9 Å (dTTP) showed clear density around the N2-ethyl-Gua lesion. The dCTP ternary structure showed good electron density for the incoming nucleotide. Electron density for incoming dTTP could not be seen except for the γ phosphate, which was included in the model. Models were built in COOT (32Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22480) Google Scholar) and refined in REFMAC5 using translesion systhesis refinement (33Winn M.D. Murshudov G.N. Papiz M.Z. Methods Enzymol. 2003; 374: 300-321Crossref PubMed Scopus (668) Google Scholar, 34Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13702) Google Scholar). The refined model converged to an Rcryst = 23.2% and Rfree = 28.6% for the dCTP-containing complex and Rcryst = 23.6% and Rfree = 28.2% for the dTTP-containing complex. Ramachandran plots for the refined models show good stereochemistry, with 87.4 (dCTP-containing) and 88.6% (dTTP-containing) of residues in the favored regions and 0.0 (dCTP-containing) and 0.0% (dTTP-containing) in the disallowed regions. Figures were prepared using PyMol (35Delano W.L. PyMol. 2002; (Delano Scientific, Palo Alto, CA)Google Scholar). Primer Extension Reactions—DNA polymerase ι catalyzes bypass of the N2-ethyl-Gua adduct using Mg2+ or Mn2+ as the activating divalent metal ion. DNA polymerases can utilize various activating divalent metals (22Beese L.S. Friedman J.M. Steitz T.A. Biochemistry. 1993; 32: 14095-14101Crossref PubMed Scopus (169) Google Scholar, 23Beese L.S. Steitz T.A. EMBO J. 1991; 10: 25-33Crossref PubMed Scopus (909) Google Scholar, 24Yang W. Lee J.Y. Nowotny M. Mol. Cell. 2006; 22: 5-13Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 25Garcia-Diaz M. Bebenek K. Krahn J.M. Pedersen L.C. Kunkel T.A. DNA Repair (Amst.). 2007; 6: 1333-1340Crossref PubMed Scopus (55) Google Scholar, 28Frank E.G. Woodgate R. J. Biol. Chem. 2007; 282: 24689-24696Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and recent evidence indicates that both Mg2+ and Mn2+ are potent activators of DNA pol ι with perhaps Mn2+ being the preferred metal ion for activation (28Frank E.G. Woodgate R. J. Biol. Chem. 2007; 282: 24689-24696Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The DNA pol ι catalyzed bypass of N2-ethyl-Gua was tested using Mg2+ or Mn2+ as the activating metal (Fig. 1). A 12-mer DNA primer was annealed to 32-mer DNA templates with the primer 3′ terminus positioned three nucleotides from the target N2-ethyl-Gua or Gua (Fig. 1A). Upon incubation of the primer-template with DNA pol ι the 12-mer primer is extended to generate products 13 to 19 nucleotides in length and no full-length 25-nucleotide products are observed (Fig. 1, B and C). These data are consistent with the previously described poor primer extension properties of DNA pol ι, which exhibits especially low efficiency when copying template pyrimidines like those positioned 5′ to the target site in this template design (18Johnson R.E. Washington M.T. Haracska L. Prakash S. Prakash L. Nature. 2000; 406: 1015-1019Crossref PubMed Scopus (573) Google Scholar, 36Tissier A. McDonald J.P. Frank E.G. Woodgate R. Genes Dev. 2000; 14: 1642-1650PubMed Google Scholar, 37Zhang Y. Yuan F. Wu X. Wang Z. Mol. Cell. Biol. 2000; 20: 7099-7108Crossref PubMed Scopus (188) Google Scholar). Primer extension reactions performed in the presence of increased concentrations of MgCl2 (Fig. 1B) or MnCl2 (Fig. 1C) show that DNA pol ι exhibits considerable sensitivity to the divalent ion concentration as indicated by the observed products. The maximum level of primer extension was detected using either DNA template at 2 mm MgCl2 (Fig. 1B) and 0.075 mm MnCl2 (Fig. 1C). Higher concentrations of MgCl2 and MnCl2 reduce DNA pol ι-catalyzed extension (Fig. 1, B and C, see also Ref. 28Frank E.G. Woodgate R. J. Biol. Chem. 2007; 282: 24689-24696Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). These data indicate that DNA pol ι-catalyzed primer extension using the N2-ethyl-Gua and Gua DNA templates is similar and that the maximum extension is achieved at a ∼26-fold lower concentration of Mn2+ ion compared with Mg2+. Insertion of the correct dCMP and incorrect dTMP opposite Gua and N2-ethyl-Gua by DNA pol ι is detected using either Mg2+ or Mn2+ as the activating metal. The 15-nucleotide products are detected as a doublet band corresponding to the correct incorporation of two dTMP nucleotides opposite the template adenines to generate the 14-nucleotide products and subsequent incorporation of dCMP or dTMP opposite the target Gua and N2-ethyl-Gua by DNA pol ι (Fig. 1, B and C). The product band corresponding to dCMP insertion opposite template Gua, but not template N2-ethyl-Gua, was detected at the higher MgCl2 concentrations tested suggesting that DNA pol ι extends the N2-ethyl-Gua:Cyt base pair more efficiently than the normal Gua:Cyt base pair when Mg2+ is the activating metal ion (Fig. 1B, compare lanes 7 and 8 to 15 and 16). The presence of the 15-nucleotide product band corresponding to insertion of dTMP opposite the target Gua and N2-ethyl-Gua in the most active primer extension reactions indicates that DNA pol ι extends more efficiently from the correctly base paired Gua:Cyt and N2-ethyl-Gua:Cyt 3′ termini relative to extension from the mispaired Gua:Thy and N2-ethyl-Gua:Thy termini (Fig. 1, B and C). The triplet band corresponding to the 16-nucleotide position indicates additional heterogeneity in the oligonucleotide product 3′ terminal sequence likely resulting from the low level of nucleotide discrimination by DNA pol ι during nucleotide incorporation opposite template cytosines (37Zhang Y. Yuan F. Wu X. Wang Z. Mol. Cell. Biol. 2000; 20: 7099-7108Crossref PubMed Scopus (188) Google Scholar). Efficiency and Fidelity of N2-Ethyl-Gua Bypass by DNA Pol ι—A steady state kinetic assay was used to more precisely quantify the efficiency and fidelity of DNA pol ι bypass of N2-ethyl-Gua compared with Gua in the presence of MgCl2 or MnCl2. Nucleotide insertion reactions were performed in the presence of increased concentrations of dCTP or dTTP using primed templates with the 3′ terminus positioned one nucleotide before the N2-ethyl-Gua or Gua. Extension reactions were performed in the presence of increased concentrations of the next correct nucleotide dGTP using primed templates with the 3′ Cyt or Thy positioned opposite the N2-ethyl-Gua or Gua. These data were quantified and the summary presented in Tables 1 and 2.TABLE 1Nucleotide insertion opposite Gua and N2-ethyl-Gua by DNA pol ι Km and kcat values were determined by quantifying gel band intensities using ImageQuant, and non-linear regression analysis of product versus [dNTP] curves, using SigmaPlot 8.0.2.Metal iondNTPKmkcatkcat/KmRelative insertion frequencyaRelative insertion frequency is calculated as 1/([kcat/Km, correct]/[kcat/Km, incorrect])μmmin–1min–1 μm–1At template N2-ethyl-Gua0.075 mm Mn2+Cyt0.10 ± 0.012425 ± 154.3 × 1031Thy0.030 ± 0.014225 ± 257.5 × 1031/0.62 mm Mg2+Cyt36 ± 374 ± 122.1 × 1001Thy650 ± 180115 ± 181.8 × 10–11/12At template Gua0.075 mm Mn2+Cyt0.15 ± 0.020700 ± 404.7 × 1031Thy0.085 ± 0.020200 ± 152.4 × 1031/22 mm Mg2+Cyt49 ± 4112 ± 182.3 × 1001Thy220 ± 6050 ± 62.3 × 10–11/10a Relative insertion frequency is calculated as 1/([kcat/Km, correct]/[kcat/Km, incorrect]) Open table in a new tab TABLE 2Extension from Gua:Cyt, Gua:Thy, and N2-ethyl-Gua:Cyt, N2-ethyl-Gua:Thy base pairs by DNA pol ι Km and kcat values were determined by quantifying gel band intensities using ImageQuant, and non-linear regression analysis of product versus [dNTP] curves, using SigmaPlot 8.0.2.Metal ionBase pairKmkcatkcat/KmRelative extension frequencyaRelative extension frequency is calculated as 1/([kcat/Km, correct]/[kcat/Km, incorrect])μmmin–1min–1 μm–1At template N2-ethyl-Gua0.075 mm Mn2+N2-Et-Gua:CytbEt, ethyl0.10 ± 0.012210 ± 72.1 × 1031N2-Et-Gua:Thy0.30 ± 0.0271.0 ± 0.023.3 × 1001/6402 mm Mg2+N2-Et-Gua:Cyt40 ± 190 ± 42.3 × 1001N2-Et-Gua:Thy160 ± 400.31 ± 0.031.9 × 10–31/1180At template Gua0.075 mm Mn2+Gua:Cyt0.25 ± 0.045220 ± 108.8 × 1021Gua:Thy1.1 ± 0.091.3 ± 0.041.2 × 1001/7302 mm Mg2+Gua:Cyt200 ± 30110 ± 85.5 × 10–11Gua:Thy170 ± 300.18 ± 0.0131.1 × 10–31/500a Relative extension frequency is calculated as 1/([kcat/Km, correct]/[kcat/Km, incorrect])b Et, ethyl Open table in a new tab The DNA pol ι inserts the correct dCMP or incorrect dTMP nucleotide at high efficiency in the presence of Mn2+. The efficiency (kcat/Km) of dCMP insertion by DNA pol ι opposite N2-ethyl-Gua in the presence of Mn2+ is ∼2,000-fold higher than that measured in the presence of Mg2+, and there is a similar high efficiency for dCMP insertion opposite Gua in the presence of Mn2+ compared with Mg2+. The dramatically higher efficiency measured in the presence of Mn2+ can be attributed to a ∼340-fold lower Km value and a ∼6-fold higher kcat value during correct nucleotide incorporation for both DNA templates (Table 1). The efficiency of incorrect dTMP insertion by DNA pol ι opposite N2-ethyl-Gua in the presence of Mn2+ is ∼42,000-fold higher than that in the presence of Mg2+ and ∼10,000-fold higher opposite Gua using Mn2+ compared with Mg2+. Similar to that observed during correct nucleotide insertion, the higher efficiency for incorrect nucleotide insertion measured in the presence of Mn2+ is mostly attributable to a much lower Km value for the nucleotide using both DNA templates. These data suggest that DNA pol ι binds correct and incorrect incoming nucleotides with greater affinity and catalyzes nucleotide addition more rapidly in the Mn2+-activated reaction compared with the Mg2+-activated reaction. The DNA pol ι exhibits higher fidelity of nucleotide insertion opposite N2-ethyl-Gua and Gua when activated with Mg2+ compared with Mn2+. Relative insertion frequencies calculated for incorrect dTMP opposite N2-ethyl-Gua and Gua in the presence of Mg2+ are lower compared with those measured using Mn2+ indicating an increased level of fidelity in the presence of Mg2+. Of particular note is the 20-fold higher level of nucleotide discrimination observed opposite N2-ethyl-Gua in the presence of Mg2+ compared with Mn2+ indicating a metal-dependent increase in the level of nucleotide discrimination opposite the adducted N2-ethyl-Gua by DNA pol ι. These data are similar to previous observations that Mn2+ causes a decreased fidelity in the replicative DNA polymerases (26Sirover M.A. Loeb L.A. Science. 1976; 194: 1434-1436Crossref PubMed Scopus (364) Google Scholar, 27Sirover M.A. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2331-2335Crossref PubMed Scop
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