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• W2024775042 abstract "The 2,4-difluorotoluene (DFT) analog of thymine has been used extensively to probe the relative importance of shape and hydrogen bonding for correct nucleotide insertion by DNA polymerases. As far as high fidelity (A-class) polymerases are concerned, shape is considered by some as key to incorporation of A(T) opposite T(A) and G(C) opposite C(G). We have carried out a detailed kinetic analysis of in vitro primer extension opposite DFT-containing templates by the trans-lesion (Y-class) DNA polymerase Dpo4 from Sulfolobus solfataricus. Although full-length product formation was observed, steady-state kinetic data show that dATP insertion opposite DFT is greatly inhibited relative to insertion opposite T (∼5,000-fold). No products were observed in the pre-steady-state. Furthermore, it is noteworthy that Dpo4 strongly prefers dATP opposite DFT over dGTP (∼200-fold) and that the polymerase is able to extend an A:DFT but not a G:DFT pair. We present crystal structures of Dpo4 in complex with DNA duplexes containing the DFT analog, the first for any DNA polymerase. In the structures, template-DFT is either positioned opposite primer-A or -G at the -1 site or is unopposed by a primer base and followed by a dGTP:A mismatch pair at the active site, representative of a -1 frameshift. The three structures provide insight into the discrimination by Dpo4 between dATP and dGTP opposite DFT and its inability to extend beyond a G:DFT pair. Although hydrogen bonding is clearly important for error-free replication by this Y-class DNA polymerase, our work demonstrates that Dpo4 also relies on shape and electrostatics to distinguish between correct and incorrect incoming nucleotide. The 2,4-difluorotoluene (DFT) analog of thymine has been used extensively to probe the relative importance of shape and hydrogen bonding for correct nucleotide insertion by DNA polymerases. As far as high fidelity (A-class) polymerases are concerned, shape is considered by some as key to incorporation of A(T) opposite T(A) and G(C) opposite C(G). We have carried out a detailed kinetic analysis of in vitro primer extension opposite DFT-containing templates by the trans-lesion (Y-class) DNA polymerase Dpo4 from Sulfolobus solfataricus. Although full-length product formation was observed, steady-state kinetic data show that dATP insertion opposite DFT is greatly inhibited relative to insertion opposite T (∼5,000-fold). No products were observed in the pre-steady-state. Furthermore, it is noteworthy that Dpo4 strongly prefers dATP opposite DFT over dGTP (∼200-fold) and that the polymerase is able to extend an A:DFT but not a G:DFT pair. We present crystal structures of Dpo4 in complex with DNA duplexes containing the DFT analog, the first for any DNA polymerase. In the structures, template-DFT is either positioned opposite primer-A or -G at the -1 site or is unopposed by a primer base and followed by a dGTP:A mismatch pair at the active site, representative of a -1 frameshift. The three structures provide insight into the discrimination by Dpo4 between dATP and dGTP opposite DFT and its inability to extend beyond a G:DFT pair. Although hydrogen bonding is clearly important for error-free replication by this Y-class DNA polymerase, our work demonstrates that Dpo4 also relies on shape and electrostatics to distinguish between correct and incorrect incoming nucleotide. Recent research on in vitro primer extension reactions catalyzed by a range of DNA polymerases and using the hydrophobic T isostere 2,4-difluorotoluene (DFT) 3The abbreviations used are:DFT2,4-difluorotolueneDpo4DNA polymerase IVCIDcollision-induced dissociationDTTdithiothreitolESIelectrospray ionizationLCliquid chromatographyMSmass spectrometryMS/MStandem mass spectrometryO6-MeGO6-methyl-GWCWatson-CrickBSAbovine serum albuminPDBProtein Data BankpolpolymeraseNSLSNational Synchrotron Light Sourcentnucleotide. 3The abbreviations used are:DFT2,4-difluorotolueneDpo4DNA polymerase IVCIDcollision-induced dissociationDTTdithiothreitolESIelectrospray ionizationLCliquid chromatographyMSmass spectrometryMS/MStandem mass spectrometryO6-MeGO6-methyl-GWCWatson-CrickBSAbovine serum albuminPDBProtein Data BankpolpolymeraseNSLSNational Synchrotron Light Sourcentnucleotide. (Fig. 1) and other analogs with substituents of increasing size at the 2- and 4-positions of the aromatic moiety appear to support different mechanisms of nucleotide insertion by high fidelity (A-class) and trans-lesion (Y-class) DNA polymerases (reviewed in Refs. 1Kool E.T. Annu. Rev. Biochem. 2002; 71: 191-219Crossref PubMed Scopus (333) Google Scholar and 2Kool E.T. Sintim H.O. Chem. Comm. 2006; : 3665-3675Crossref PubMed Google Scholar). Thus, accurate replication by A-class polymerases may be more dependent on a close steric match between the active site and the shape of a Watson-Crick base pair (“active site tightness”) than on the formation of complementary hydrogen bonds between incoming nucleotide base and template residue (3Morales J.C. Kool E.T. Nat. Struct. Biol. 1998; 5: 950-954Crossref PubMed Scopus (264) Google Scholar, 4Kim T.W. Delaney J.C. Essigmann J.M. Kool E.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15803-15808Crossref PubMed Scopus (105) Google Scholar, 5Potapova O. Chan C. DeLucia A.M. Helquist S.A. Kool E.T. Grindley N.D.F. Joyce C.M. Biochemistry. 2006; 45: 890-898Crossref PubMed Scopus (37) Google Scholar, 6Kim T.W. Brieba L.G. Ellenberger T. Kool E.T. J. Biol. Chem. 2006; 281: 2289-2295Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Conversely, their more open active sites may render Y-class polymerases (6Kim T.W. Brieba L.G. Ellenberger T. Kool E.T. J. Biol. Chem. 2006; 281: 2289-2295Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 7Washington M.T. Helquist S.A. Kool E.T. Prakash L. Prakash S. Mol. Cell. Biol. 2003; 23: 5107-5112Crossref PubMed Scopus (80) Google Scholar, 8Wolfle W.T. Washington M.T. Kool E.T. Spratt T.E. Helquist S.A. Prakash L. Prakash S. Mol. Cell. Biol. 2005; 25: 7137-7143Crossref PubMed Scopus (50) Google Scholar, 9Mizukami S. Kim T.W. Helquist S.A. Kool E.T. Biochemistry. 2006; 45: 2772-2778Crossref PubMed Scopus (70) Google Scholar, 10Boudsocq F. Iwai S. Hanaoka F. Woodgate R. Nucleic Acids Res. 2001; 29: 4607-4616Crossref PubMed Google Scholar) less sensitive to changes in base pair dimensions but more dependent on formation of hydrogen bonds between nucleotide pairs at the replicative position (6Kim T.W. Brieba L.G. Ellenberger T. Kool E.T. J. Biol. Chem. 2006; 281: 2289-2295Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 7Washington M.T. Helquist S.A. Kool E.T. Prakash L. Prakash S. Mol. Cell. Biol. 2003; 23: 5107-5112Crossref PubMed Scopus (80) Google Scholar, 8Wolfle W.T. Washington M.T. Kool E.T. Spratt T.E. Helquist S.A. Prakash L. Prakash S. Mol. Cell. Biol. 2005; 25: 7137-7143Crossref PubMed Scopus (50) Google Scholar, 9Mizukami S. Kim T.W. Helquist S.A. Kool E.T. Biochemistry. 2006; 45: 2772-2778Crossref PubMed Scopus (70) Google Scholar). A-class DNA polymerases whose activities were assessed using apolar T analogs to date include Escherichia coli pol I (3Morales J.C. Kool E.T. Nat. Struct. Biol. 1998; 5: 950-954Crossref PubMed Scopus (264) Google Scholar, 4Kim T.W. Delaney J.C. Essigmann J.M. Kool E.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15803-15808Crossref PubMed Scopus (105) Google Scholar, 5Potapova O. Chan C. DeLucia A.M. Helquist S.A. Kool E.T. Grindley N.D.F. Joyce C.M. Biochemistry. 2006; 45: 890-898Crossref PubMed Scopus (37) Google Scholar) and the polymerase from phage T7 (6Kim T.W. Brieba L.G. Ellenberger T. Kool E.T. J. Biol. Chem. 2006; 281: 2289-2295Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and the tested Y-class DNA polymerases consisting of yeast pol η (7Washington M.T. Helquist S.A. Kool E.T. Prakash L. Prakash S. Mol. Cell. Biol. 2003; 23: 5107-5112Crossref PubMed Scopus (80) Google Scholar), human pol κ (8Wolfle W.T. Washington M.T. Kool E.T. Spratt T.E. Helquist S.A. Prakash L. Prakash S. Mol. Cell. Biol. 2005; 25: 7137-7143Crossref PubMed Scopus (50) Google Scholar), and the Dbh (DinB homolog (5Potapova O. Chan C. DeLucia A.M. Helquist S.A. Kool E.T. Grindley N.D.F. Joyce C.M. Biochemistry. 2006; 45: 890-898Crossref PubMed Scopus (37) Google Scholar)) and Dpo4 (9Mizukami S. Kim T.W. Helquist S.A. Kool E.T. Biochemistry. 2006; 45: 2772-2778Crossref PubMed Scopus (70) Google Scholar) polymerases from Sulfolobus acidocaldarius and Sulfolobus solfataricus, respectively.Careful review of the accumulated data regarding A-class polymerases (i.e. E. coli DNA pol I Klenow fragment) also reveals that in contrast to the relatively modest reduction in efficiency for inserting dATP opposite DFT, the efficiencies of extension after pairs comprising the hydrophobic analog are drastically inhibited (5Potapova O. Chan C. DeLucia A.M. Helquist S.A. Kool E.T. Grindley N.D.F. Joyce C.M. Biochemistry. 2006; 45: 890-898Crossref PubMed Scopus (37) Google Scholar). Moreover, there is a significant asymmetry in the efficiency of the extension reaction, an ∼30-fold reduction for insertion of dATP opposite template DFT and ∼900-fold reduction for insertion of d(DFT)TP opposite template A (relative to the corresponding processes with T and dTTP, respectively (5Potapova O. Chan C. DeLucia A.M. Helquist S.A. Kool E.T. Grindley N.D.F. Joyce C.M. Biochemistry. 2006; 45: 890-898Crossref PubMed Scopus (37) Google Scholar)). The latter loss is comparable with the effects on insertion reactions involving the DFT analog seen with Y-class polymerases (5Potapova O. Chan C. DeLucia A.M. Helquist S.A. Kool E.T. Grindley N.D.F. Joyce C.M. Biochemistry. 2006; 45: 890-898Crossref PubMed Scopus (37) Google Scholar, 7Washington M.T. Helquist S.A. Kool E.T. Prakash L. Prakash S. Mol. Cell. Biol. 2003; 23: 5107-5112Crossref PubMed Scopus (80) Google Scholar, 8Wolfle W.T. Washington M.T. Kool E.T. Spratt T.E. Helquist S.A. Prakash L. Prakash S. Mol. Cell. Biol. 2005; 25: 7137-7143Crossref PubMed Scopus (50) Google Scholar, 9Mizukami S. Kim T.W. Helquist S.A. Kool E.T. Biochemistry. 2006; 45: 2772-2778Crossref PubMed Scopus (70) Google Scholar). Together, these observations appear inconsistent with the conclusion that A- and Y-class polymerases use different mechanisms of replication and that the former may rely chiefly on shape for efficient and correct nucleotide insertion (1Kool E.T. Annu. Rev. Biochem. 2002; 71: 191-219Crossref PubMed Scopus (333) Google Scholar, 2Kool E.T. Sintim H.O. Chem. Comm. 2006; : 3665-3675Crossref PubMed Google Scholar). An important limitation of the large body of work involving the use of the hydrophobic T isostere DFT for probing enzyme mechanism and the role of hydrogen bonding in DNA replication is constituted by the fact that the analog has never been visualized at the active site of any DNA polymerase.The Dpo4 trans-lesion DNA polymerase from S. solfataricus (10Boudsocq F. Iwai S. Hanaoka F. Woodgate R. Nucleic Acids Res. 2001; 29: 4607-4616Crossref PubMed Google Scholar) has been investigated in more detail both in terms of its function (10Boudsocq F. Iwai S. Hanaoka F. Woodgate R. Nucleic Acids Res. 2001; 29: 4607-4616Crossref PubMed Google Scholar, 11Fiala K.A. Suo Z. Biochemistry. 2004; 43: 2116-2125Crossref PubMed Scopus (109) Google Scholar, 12Vaisman A. Ling H. Woodgate R. Yang W. EMBO J. 2005; 24: 2957-2967Crossref PubMed Scopus (157) Google Scholar, 13Irimia A. Zang H. Loukachevitch L.V. Guengerich F.P. Egli M. Biochemistry. 2006; 45: 5949-5956Crossref PubMed Scopus (28) Google Scholar, 14Guengerich F.P. Chem. Rev. 2006; 106: 420-452Crossref PubMed Scopus (94) Google Scholar) and structure (15Ling H. Boudsocq F. Woodgate R. Yang W. Cell. 2001; 107: 91-102Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar, 16Trincao J. Johnson R.E. Wolfle W.T. Escalante C.R. Prakash S. Prakash L. Aggarwal A.K. Nat. Struct. Mol. Biol. 2004; 11: 457-462Crossref PubMed Scopus (65) Google Scholar, 17Ling H. Boudsocq F. Woodgate R. Yang W. Mol. Cell. 2004; 13: 751-762Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) than any other representatives of the Y-class family of polymerases. Numerous crystal structures of Dpo4 in complex with DNA template-primer constructs containing adducted nucleotides have been determined during the last 4 years (18Ling H. Boudsocq F. Plosky B.S. Woodgate R. Yang W. Nature. 2003; 424: 1083-1087Crossref PubMed Scopus (202) Google Scholar, 19Ling H. Sayer J.M. Plosky B.S. Yagi H. Boudsocq F. Woodgate R. Jerina D.M. Yang W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2265-2269Crossref PubMed Scopus (161) Google Scholar, 20Zang H. Goodenough A.K. Choi J.-Y. Irimia A. Loukachevitch L.V. Kozekov I.D. Angel K.C. Rizzo C.J. Egli M. Guengerich F.P. J. Biol. Chem. 2005; 280: 29750-29764Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 21Zang H. Irimia A. Choi J.-Y. Angel K.C. Loukachevitch L.V. Egli M. Guengerich F.P. J. Biol. Chem. 2006; 281: 2358-2372Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 22Eoff R.L. Irimia A. Egli M. Guengerich F.P. J. Biol. Chem. 2007; 282: 1456-1467Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 23Eoff R.L. Angel K.C. Egli M. Guengerich F.P. J. Biol. Chem. 2007; 282: 13573-13584Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 24Eoff R.L. Irimia A. Angel K.C. Egli M. Guengerich F.P. J. Biol. Chem. 2007; 282: 19831-19843Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The structural data reveal a versatile enzyme that in some cases defies the view of Y-class polymerases as low processivity and/or fidelity catalysts of DNA replication. For example, Dpo4 was found to synthesize past the 8-oxoG adduct efficiently and with relatively good fidelity, incorporating ≥95% dCTP instead of dATP and exhibiting faster rates for pre-steady-state kinetics of dCTP incorporation opposite 8-oxoG than G (21Zang H. Irimia A. Choi J.-Y. Angel K.C. Loukachevitch L.V. Egli M. Guengerich F.P. J. Biol. Chem. 2006; 281: 2358-2372Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). As far as the different roles of sterics versus hydrogen bonding in the replications catalyzed by A-class and Y-class DNA polymerases are concerned, recent crystal structures of a the high fidelity Bacillus stearothermophilus DNA polymerase I large fragment (BF) and Dpo4 in complex with DNAs containing the O6-methyl-G (O6-MeG) adduct opposite C and T may be instructive (see Refs. 25Warren J.J. Forsberg L.J. Beese L.S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 19701-19706Crossref PubMed Scopus (115) Google Scholar and 22Eoff R.L. Irimia A. Egli M. Guengerich F.P. J. Biol. Chem. 2007; 282: 1456-1467Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, respectively). At the constrained active site of BF, both the O6-MeG:C and O6-MeG:T pairs adopt a Watson-Crick conformation, whereby the cytosine in the former is protonated. This finding is consistent with the preferential incorporation of T over C opposite O6-MeG by high fidelity polymerases. The Watson-Crick mode for correctly and mispaired combinations was also observed at the -1- and -2-bp positions. On the other hand, the O6-MeG:C pair at the more spacious active site of Dpo4 adopts a wobble geometry, and analysis of Dpo4-catalyzed extension products reveals that the enzyme accurately bypasses O6-MeG with C being the major product (>70%) and T and A being the minor species (22Eoff R.L. Irimia A. Egli M. Guengerich F.P. J. Biol. Chem. 2007; 282: 1456-1467Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). T exhibited multiple conformations opposite O6-MeG at the Dpo4 active site that most likely include both the Watson-Crick and wobble geometries.Here we report the results of detailed pre-steady-state and steady-state kinetic analyses as well as LC-MS/MS characterizations (20Zang H. Goodenough A.K. Choi J.-Y. Irimia A. Loukachevitch L.V. Kozekov I.D. Angel K.C. Rizzo C.J. Egli M. Guengerich F.P. J. Biol. Chem. 2005; 280: 29750-29764Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 21Zang H. Irimia A. Choi J.-Y. Angel K.C. Loukachevitch L.V. Egli M. Guengerich F.P. J. Biol. Chem. 2006; 281: 2358-2372Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) of full-length products of Dpo4-catalyzed primer extension reactions opposite template strands containing DFT either at the replicative or -1-bp positions. Our steady-state kinetic data confirm that the catalytic efficiency of Dpo4-catalyzed insertion opposite DFT was strongly inhibited, consistent with previously published data by others (9Mizukami S. Kim T.W. Helquist S.A. Kool E.T. Biochemistry. 2006; 45: 2772-2778Crossref PubMed Scopus (70) Google Scholar). However, it is noteworthy that Dpo4 is ∼200-fold more efficient (kcat/Km) at inserting dATP opposite DFT than dGTP. The pre-steady-state kinetic analysis revealed no product formation for insertion of dATP opposite DFT, and next-base extension reactions were slower following an A:DFT pair and prevented by a G:DFT pair. To interpret the activity data, we determined crystal structures of Dpo4 in complex with primer-template DNA duplexes featuring dGTP opposite DFT at the active site as well as G or A paired with DFT at the -1 position. The structures provide insight into the different efficiencies of Dpo4 for inserting A and G opposite template-DFT and allow a rationalization of the inability of Dpo4 to further extend the primer strand after a G:DFT pair. The structure featuring an A:DFT pair also challenges the usual assumption that this pair virtually matches A:T in shape. Thus, the absence of hydrogen bonds in the former actually leads to significant changes in the local geometry of the template-primer duplex. Although Dpo4 may rely more heavily on hydrogen bonds between incoming nucleotide and template base for error-free replication compared with high fidelity polymerases, sterics clearly play a role in discriminating between A and G as the pairing partner of DFT.EXPERIMENTAL PROCEDURESMaterials—Dpo4 was expressed in E. coli and purified to electrophoretic homogeneity as described previously (20Zang H. Goodenough A.K. Choi J.-Y. Irimia A. Loukachevitch L.V. Kozekov I.D. Angel K.C. Rizzo C.J. Egli M. Guengerich F.P. J. Biol. Chem. 2005; 280: 29750-29764Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). All unlabeled dNTPs were obtained from Amersham Biosciences, and [γ-32P]ATP was purchased from PerkinElmer Life Sciences. All unmodified oligonucleotides used in this work were synthesized by Integrated DNA Technologies (Coralville, IA). The DFT phosphoramidite was purchased from Glen Research (Sterling, VA), and DFT-modified template strands were synthesized using an ABI 381A oligonucleotide synthesizer on a 1 μm scale following standard solid phase synthesis and purification protocols.Full-length Extension Assay—A 32P-labeled primer was annealed to template oligonucleotide by heating a 1:1 solution of oligonucleotide to 95 °C for 5 min and then slow cooling to room temperature. The primer was then incubated with Dpo4 and extended in the presence of a mixture of all four dNTPs. Each reaction was initiated by adding dNTP·Mg2+ (1 mm of each dNTP and 5 mm MgCl2) solution to a preincubated Dpo4·DNA complex (100 nm Dpo4 and 200 nm DNA). The reaction was carried out at 37 °C in 50 mm Tris-HCl (pH 7.4), 50 mm NaCl, 5 mm DTT, 100 μg ml-1 BSA, and 5% (v/v) glycerol. At the indicated time, 5-μl aliquots were quenched with 50 μl of 500 mm EDTA (pH 9.0). The samples were then mixed with 100 μl of a 95% formamide, 20 mm EDTA solution and were separated on a 20% polyacrylamide (w/v), 7 m urea gel.Steady-state Kinetics—Dpo4-catalyzed single nucleotide incorporation was measured over a range of dNTP concentrations. All reactions were carried out at 37 °C in 50 mm Tris-HCl (pH 7.4) buffer containing 50 mm NaCl, 5.0 mm DTT, 50 μg ml-1 BSA, and 5% glycerol (v/v). Dpo4 (10 nm) was preincubated with radiolabeled DNA (100 nm), and the reaction was initiated by adding dNTP·Mg2+. Aliquots were quenched with 500 mm EDTA (pH 9.0) after varying incubation times. Substrate and product DNA were separated by electrophoresis on a 20% polyacrylamide (w/v), 7 m urea gel. The products were then visualized using a PhosphorImager and quantitated using Quantity One™ software (Bio-Rad). The initial portion of the velocity curve was fit to a linear equation in the program GraphPad Prism (GraphPad, San Diego). The resulting velocity was plotted as a function of dNTP concentration and then fit to a hyperbola, correcting for enzyme concentration, to obtain estimates of kcat and Km,dNTP (Table 1).TABLE 1Steady-state kinetic parameters for 1-base incorporation by Dpo4Oligomer pairPrimer-template pairdNTPkcatKm,dNTPΔEfficiency relative to dCTP:Gmin-1μm13-merdCTP34.8 ± 0.6aData are from Ref. 223.0 ± 0.2aData are from Ref. 2218-mer-G12-merdATP1.8 ± 0.10816 ± 785,300-Fold less18-mer-DFT12-merdTTP0.19 ± 0.041320 ± 56080,000-Fold less18-mer-DFT12-merdCTP0.15 ± 0.01440 ± 9634,000-Fold less18-mer-DFT12-merdGTP0.010 ± 0.002870 ± 430106-Fold less18-mer-DFT12-UdATP1.8 ± 0.1745 ± 314,800-Fold less18-mer-DFT12-UdTTP0.10 ± 0.01890 ± 200103,000-Fold less18-mer-DFT12-UdCTP0.27 ± 0.02550 ± 9524,000-Fold less18-mer-DFT12-UdGTP0.015 ± 0.003780 ± 340604,000-Fold less18-mer-DFTa Data are from Ref. 22Eoff R.L. Irimia A. Egli M. Guengerich F.P. J. Biol. Chem. 2007; 282: 1456-1467Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Open table in a new tab Transient-state Kinetics—All pre-steady-state experiments were performed using a KinTek RQF-3 model chemical quench-flow apparatus (KinTek Corp., Austin, TX) with 50 mm Tris-HCl buffer (pH 7.4) in the drive syringes. All RQF experiments were carried out at 37 °C in a buffer containing 50 mm Tris-HCl buffer (pH 7.4) containing 50 mm NaCl, 5 mm DTT, 100 μg ml-1 BSA, and 5% (v/v) glycerol. Polymerase catalysis was stopped by the addition of 500 mm EDTA (pH 9.0). Substrate and product DNA was separated by electrophoresis on a 20% polyacrylamide (w/v), 7 m urea gel. The products were then visualized using a PhosphorImager and quantitated using Quantity One™ software. Results obtained under single-turnover conditions were fit to Equation 1, y=A(1-e-kobst)(Eq. 1) where A is product formed in first binding event; kobs is rate constant defining polymerization under the conditions used for the experiment being analyzed, and t indicates time.LC-MS/MS Analysis of Oligonucleotide Products from Dpo4 Reactions—Dpo4 (5 μm) was preincubated with primer-template DNA (10 μm), and the reaction was initiated by addition of dNTP (1 mm each) and MgCl2 (5 mm) in a final volume of 100 μl. Dpo4 catalysis was allowed to proceed at 37 °C for 4 h in 50 mm Tris-HCl buffer (pH 7.8 at 25 °C) containing 50 mm NaCl, 1 mm DTT, 50 μg ml-1 BSA, and 5% glycerol (v/v). The reaction was terminated by extraction of the remaining dNTPs using a size-exclusion chromatography column (Bio-Spin 6 chromatography column, Bio-Rad). Concentrated stocks of Tris-HCl, DTT, and EDTA were added to restore the concentrations to 50, 5, and 1 mm, respectively. Next, E. coli uracil DNA glycosylase (20 units, Sigma) was added, and the solution was incubated at 37 °C for 6 h to hydrolyze the uracil residue on the extended primer. The reaction mixture was then heated at 95°C for 1 h in the presence of 0.25 m piperidine, followed by removal of the solvent by centrifugation under vacuum. The dried sample was resuspended in 100 μl of H2O for MS analysis.LC-MS/MS analysis (20Zang H. Goodenough A.K. Choi J.-Y. Irimia A. Loukachevitch L.V. Kozekov I.D. Angel K.C. Rizzo C.J. Egli M. Guengerich F.P. J. Biol. Chem. 2005; 280: 29750-29764Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 21Zang H. Irimia A. Choi J.-Y. Angel K.C. Loukachevitch L.V. Egli M. Guengerich F.P. J. Biol. Chem. 2006; 281: 2358-2372Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) was performed on a Waters Acquity UPLC system (Waters) connected to a Finnigan LTQ mass spectrometer (Fisher), operating in the ESI negative ion mode (Table 2). An Acquity UPLC BEH octadecylsilane (C18) column (1.7 μm, 1.0 × 100 mm) was used with the following LC conditions: Buffer A contained 10 mm NH4CH3CO2 plus 2% CH3CN (v/v), and Buffer B contained 10 mm NH4CH3CO2 plus 95% CH3CN (v/v). The following gradient program was used with a flow rate of 150 μl min-1: 0-2.5 min, linear gradient from 100% A to 95% A, 5% B (v/v); 2.5-6.0 min, linear gradient to 75% A, 25% B (v/v); 6-6.5 min, linear gradient to 100% B; 6.5-8.0 min, hold at 100% B; 8.0-9.0 min, linear gradient to 100% A; 9.0-12.0 min, hold at 100% A. The temperature of the column was maintained at 50 °C. Samples were injected with an autosampler system. ESI conditions were as follows: source voltage 4 kV; source current 100 μA; auxiliary gas flow rate setting 20; sweep gas flow rate setting 5; sheath gas flow setting 34; capillary voltage -49 V; capillary temperature 350 °C; tube lens voltage -90 V. MS/MS conditions were as follows: normalized collision energy 35%; activation Q 0.250; activation time 30 ms. The doubly (negatively) charged species were generally used for CID analysis. The calculations of the CID fragmentations of oligonucleotide sequences were done using a program linked to the Mass Spectrometry Group of Medicinal Chemistry at the University of Utah. The nomenclature used in supplemental Tables S1-S6 has been described previously (26Christian N.P. Reilly J.P. Mokler V.R. Wincott F.E. Ellington A.D. J. Am. Soc. Mass. Spec. 2001; 12: 744-753Crossref PubMed Scopus (25) Google Scholar).TABLE 2Results of LC-MS analysis of Dpo4-catalyzed full-length extension productsProduct% of total5′-pACTGAA-3′645′-pACTGAAA-3′105′-pCCTGAA-3′85′-pTCTGAA-3′55′-pGCTGAA-3′35′-pCTGAA-3′10 Open table in a new tab Dpo4 Pyrophosphorolysis Activity—The effect of DFT upon the pyrophosphorolysis activity of Dpo4 was tested by incubating Dpo4 (100 nm) with a radiolabeled DNA substrate (100 nm) in 50 mm Tris-HCl buffer (pH 7.4) containing 50 mm NaCl, 10.0 mm DTT, 200 μg ml-1 BSA, and 2.5% glycerol (v/v) at 60 °C. Two DNA templates were used, a control 18-mer template containing T and the 18-mer containing DFT. A 13-mer primer containing A at the 3′ terminus was annealed to the template DNA so that it paired with T or DFT. Pyrophosphorolysis was initiated by addition of sodium pyrophosphate (pH 7.4; 500 μm final concentration). Aliquots were quenched with 20 mm EDTA (pH 9.0) in 95% formamide (v/v) after varying incubation times. Substrate and product DNA were separated and imaged as described for the steady-state assays. The initial portion of the velocity curve was fit to a linear equation in the program GraphPad Prism.Crystallization and X-ray Diffraction Data Collection—The 18-nt template 5′-TTCAG(DFT)AGTCCTTCCCCC-3′ was annealed with the 13-nt primer 5′-GGGGGAAGGACTX-3′, with X either G in the Dpo4(DFT:13G) complex or A in the Dpo4(DFT:13A) complex. For the insertion complex Dpo4(dGTP), the 18-nt template 5′-TTCA(DFT)TAGTCCTTCCCCC-3′ was annealed with the 13-nt primer 5′-GGGGGAAGGACTA-3′. The Dpo4 protein was mixed with the DNA duplex in a buffer containing 60 mm NaCl, 4% glycerol, 20 mm Tris (pH 7.4), 5 mm CaCl2, and 1 mm d(d)NTP (either dGTP or ddCTP, see Table 3). Droplets consisted of a 1:1 mixture of the protein·DNA complex and reservoir solutions. In the case of the Dpo4(DFT:13A) complex crystals were obtained by equilibrating droplets against reservoir solution containing 12% polyethylene glycol 3350, 0.2 m ammonium acetate, 0.1 m calcium acetate, and 20 mm Tris (pH 7.5). Crystals of the Dpo4(DFT:13G) and Dpo4(dGTP) complexes were grown from droplets equilibrated against reservoir solutions containing 16-24% polyethylene glycol 3350, 0.1 m Ca(OAc)2, and 20 mm Tris (pH 7.5).TABLE 3Selected crystal data, data collection, and refinement parametersParameterDpo4(DFT:13A)Dpo4(dGTP)Dpo4(DFT:13G)Type of complexPostinsertionInsertionPostinsertionCrystal data, data collection X-ray sourceAPS(DND-CAT)NSLSAPS(SER-CAT) BeamlineID-5X29ID-22 DetectorMARCCDQuantum CCDMARCCD Wavelength (Å)1.001.001.00 Temperature (K)110110110 No. of crystals111 Space groupP21P21212P21 Unit cell (a, b, and c; Å)52.14,101.87,111.0694.62,103.56,52.6452.7,186.74,52.73 (α, β, and γ; °)90,94.9,9090,90,9090,110.1,90 Resolution range (Å)50.0-2.850.0-2.9850.0-3.0 Highest resolution shellaValues in parentheses correspond to the highest resolution shells(2.9-2.8)(3.09-2.98)(3.19-3.0) No. of measurements113,315 (10,980)31,795 (1,726)61,192 (3,171) No. of unique reflections27,671 (2,611)9,628 (863)16,865 (1,510) Redundancy4.1 (4.2)3.3 (2.0)3.6 (2.1) Completeness (%)96.6 (91.2)84.5 (81.0)87.5 (81.0) RmergebRmerge = Σhkl Σj= 1,N|〈Ihkl〉 - Ihklj|/Σhkl Σj= 1,N|Ihklj|, where the outer sum (hkl) is taken over the unique reflections7.0 (40.1)9.1 (30.0)14.2 (52.5) Signal to noise (〈I/σI〉)16.3 (3.42)14.3 (4.05)9.2 (2.0) Solvent content (%)58.954.252.05Refinement Model composition (asymmetric unit)No. of amino acid residues342/343342343/342No. of water molecules21666159No. of Ca2+ ions3/332/2No. of template nucleotides16/181618/16No. of primer nucleotides13/131313/13No. of dGTP1No. of ddCTP1/11/1RfcRf = Σhkl||Fo,hkl| - k|Fc,hkl||/Σhkl|Fo,hkl|, where |Fo,hkl| and |Fc,hkl| are the observed and calculated structure factor amplitudes, respectively (%)21.123.021.8RfreedRfree idem, for the set of reflections (5% of the total) omitted from the refinement process (%)24.928.229.0Estimated coordinate error (Å) Luzatti plot0.360.380.38 Luzatti plot (c-v)0.440.530.55 sA plot0.50.530.57 σA plot (c-v)0.60.720.8" @default.
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• W2024775042 title "Structure and Activity of Y-class DNA Polymerase DPO4 from Sulfolobus solfataricus with Templates Containing the Hydrophobic Thymine Analog 2,4-Difluorotoluene" @default.
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