Lesion orientation of O4-alkylthymidine influences replication by human DNA polymerase η

Conformation of the α-carbon of O4-alkylthymidine was shown to exert an influence on human DNA polymerase η (hPol η) bypass. Crystal structures of hPol η·DNA·dNTP ternary complexes reveal a unique conformation adopted by O4-methylthymidine, where the nucleobase resides nestled at the active site ceiling where hydrogen-bonding with the incoming nucleotide is prevented.


Supplementary Methods
General methods for the preparation and characterization of nucleosides

Supplementary Methods
General methods for the preparation and characterization of nucleosides.  13 C NMR spectra ( 1 H decoupled) were recorded at a frequency of 125.7 MHz and chemical shifts were reported in ppm with tetramethylsilane as a reference. 31 P NMR spectra ( 1 H decoupled) were recorded at a frequency of 202.3 MHz and chemical shifts were reported in ppm with H 3 PO 4 used as an external standard. High resolution mass spectrometry of modified nucleosides were carried out using an LTQ Orbitrap Velos -ETD mass spectrometer (Thermo Scientific) at the Concordia University Centre for Biological Applications of Mass Spectrometry (CBAMS) or using a 7T-LTQ FT ICR mass spectrometer (Thermo Scientific) at the Concordia University Centre for Structural and Functional Genomics. The mass spectrometer was operated in full scan, positive ion detection mode. ESI mass spectra for oligonucleotides were obtained at CBAMS using a Micromass Qtof2 mass spectrometer (Waters) equipped with a nanospray ion source. The mass spectrometer was operated in full scan, negative ion detection mode.

5-Hydroxyethyluracil (2a)
To a solution of NaOEt (1.23 g, 18.1 mmol) in EtOH (90 mL) was added compound 1a (7.55 g, 48.4 mmol). After 16h, the solvent was removed in vacuo and the crude was taken up in a minimal amount of boiling water. The solution was acidified (to a pH ~ 2-3) on an ice bath and the precipitate was isolated by vacuum filtration and washed with cold EtOH (3  40 mL). To maximize the yield, the filtrate was evaporated and the solid was resuspended in EtOH (50 mL). The solid was isolated and washed once more according to the procedure described above to give 2a in an overall yield of 7.16 g (94.8%) as a colorless powder. λ max(MeCN) 262 nm. 1

5-Hydroxypropyluracil (2b)
To a solution of NaOEt (1.52 g, 22.4 mmol) in EtOH (225 mL) was added compound 1b (4.76 g, 28.0 mmol). After 16h, the solvent was removed in vacuo and the crude was taken up in a minimal amount of boiling water. The solution was acidified (to a pH ~ 2-3) and cooled, and the precipitate was isolated by vacuum filtration and washed with cold EtOH (3  50 mL). To maximize the yield, the filtrate was evaporated and the solid was resuspended in EtOH (50 mL). The solid was isolated and washed once more according to the procedure described above to give 2b in an overall yield of 4.75 g (>99%) as a colorless powder. λ max(MeCN) 261 nm. 1 To a solution of compound 2a (2.4 g, 15.4 mmol) in HMDS (60 mL, 164 mmol) was added TMS-Cl (0.4 mL, 3.14 mmol) which was then stirred vigorously at 140 o C. After 5 h the excess HMDS was removed in vacuo and the resulting gum was left on the high vacuum for 2 h. The gum was then dissolved in 1,2-dichloroethane (60 mL) and to this was added a solution of 1-(α)chloro-3,5-di-O-(p-toluoyl)-2-deoxy-D-ribose (2.0 g, 5.1 mmol) in 1,2-dichloroethane (80 mL) while stirring at room temperature. After 16 h the solvent was removed in vacuo and the residue was taken up in CH 2 Cl 2 (60 mL) then washed with 3% (aq) NaHCO 3 (w/v, 2  50 mL). The organic layer was dried over anhydrous Na 2 SO 4 , decanted and the solvent evaporated. Then, 1 M TBAF (in THF, 5 mL, 5 mmol) was added and the reaction stirred for 15 min. The solvent was removed and the mixture of anomers was purified via flash column chromatography using a CH 3 OH:CH 2 Cl 2 solvent system (2% → 5%, v/v). The β-anomer was isolated by precipitation from EtOAc: hexanes (1:1, v/v) at a concentration of 31 mM with stirring at room temperature for 10 min. The resulting suspension was filtered under vacuo to afford 1.15 g (44%) of 3a as a colorless foam. R f (SiO 2 TLC): 0.33 CH 3 OH:CH 2 Cl 2 (5%, v/v). λ max(MeCN) 241 nm. 1  was taken up in DCM (50 mL), washed with 3% (aq.) NaHCO 3 (2  50 mL), dried over anhydrous Na 2 SO 4 , decanted and the solvent evaporated. Purification was performed by flash column chromatography using an EtOAc: Hex solvent system (1:4, then 1:3, v/v) to afford 3.1 g (65%) of the β-anomer and 0.82 g (17 %) of the α-anomer, both as colorless foams. To a solution of the β-anomer 3a (2.6 g, 4.1 mmol) in THF (40 mL) was added 1 M TBAF (in THF, 4.9 mL, 4.9 mmol) which was heated to 45 o C under stirring. After 1 h the solvent was removed in vacuo and the residue was taken up in DCM (50 mL) then washed with 3% (aq., w/v) NaHCO 3 (2  50 mL). The organic layer was dried over anhydrous Na 2 SO 4 , decanted and the solvent was evaporated. Purification was achieved by flash column chromatography using a CH 3 OH:CH 2 Cl 2 solvent system (1%  3%, v/v) to afford 2.0 g (96 %) of 3a as a colorless foam.
To a solution of compound 2b (4.5 g, 26.4 mmol) in HMDS (95 mL) was added TMS-Cl (0.73 mL, 5.74 mmol) while stirred at 135 o C. After 5h, the excess HMDS was removed in vacuo and the resulting gum was placed on the high vacuum for 1 h. To a solution of the gum in 1,2dichloroethane (225 mL) was added 1-(α)-chloro-3,5-di-O-(p-toluoyl)-2-deoxy-D-ribose (3.5 g, 9.0 mmol) and the atmosphere was exchanged with Ar while stirring at room temperature. After 16h the solvent was removed in vacuo and the crude was taken up in CH 2 Cl 2 (125 mL), washed with cold brine (50 mL) followed by cold 3% (aq., w/v) NaHCO 3 (2  50mL). The organic layer was dried over anhydrous Na 2 SO 4 , decanted and the solvent was evaporated. The product was purified via flash column chromatography (short column) using an isocratic CH 3 OH:CH 2 Cl 2 solvent system (4%, v/v). A mixture of silylated and desilylated products were isolated to which 1 M TBAF (in THF, 5 mL, 5 mmol) was added under stirring. After 15 min, the solvent was removed in vacuo and the mixture of desilylated anomers were purified by flash column chromatography using a CH 2 Cl 2 :CH 3 OH solvent system (2 % → 4 % by increments of 0.5 %, v/v). The β-anomer was isolated by precipitation from EtOAc at a concentration of 31 mM with stirring at room temperature for 10 min. Alternative method for the resolution of anomers: To a solution of a purified mixture of anomers (2.0 g, 3.8 mmol) and imidazole (1.2 g, 17 mmol) in CH 2 Cl 2 (38 mL) was added TBS-Cl (1.3 g, 8.4 mmol) while stirring. After 4h the solvent was removed in vacuo and the content was taken up in CH 2 Cl 2 (50 mL) and washed with 3% (aq., w/v) NaHCO 3 (2  50 mL). The organic layer was dried over anhydrous Na 2 SO 4 , decanted and the solvent removed in vacuo. Purification was achieved by flash column chromatography using an EtOAc:hexanes solvent system (35:65, v/v) to afford 1.2 g (49 %) of the β-anomer and 0.68 g (28 %) of the α-anomer, both as colorless foams. To a solution of the β-anomer of 3b (0.50 g, 0.79 mmol) in THF (4 mL) was added 1 M TBAF (in THF, 1.2 mL, 1.2 mmol) which was heated to 45 o C with stirring. After 4 h the solvent was removed in vacuo and the content purified by flash column chromatography using a solvent system (1%  3%, v/v) to afford 0.33 g (81 %) of 3b a colorless foam.
To a solution of compound 3a (1.2 g, 2.3 mmol) in pyridine (25 mL) was added MsCl (240 μL, 3.1 mmol) dropwise at 0 o C while stirring. After 5 h, the reaction was diluted with THF (300 mL) and DBU (1.2 mL, 8.0 mmol) was added and heated to a gentle reflux. After 16 h the solvent was removed in vacuo and the content was taken up in CH 2 Cl 2 (100 mL), washed with brine (2 × 50 mL) and 3% (aq.) NaHCO 3 (2 x 50 mL). The organic layer was dried over anhydrous Na 2 SO 4 , decanted and the solvent was removed in vacuo to produce a yellow gum. The product was purified via flash column chromatography using a gradient of CH 2 To a solution of compound 3b (1.5 g, 2.9 mmol) in CH 2 Cl 2 (30 mL) was added NEt 3 (0.80 mL, 5.74 mmol) which was allowed to stir at 0 o C for 15 min. MsCl (0.30 mL, 3.9 mmol) was added dropwise over 10 min and cooled to -20 o C for 1 h without stirring. The reaction was diluted with CH 2 Cl 2 (100 mL) and washed with ice/water (50 mL), ice/brine (50 mL) and ice/3% aq. (w/v) NaHCO 3 (50 mL). The organic layer was then dried over anhydrous Na 2 SO 4 , decanted and the solvent was removed in vacuo to produce a yellow gum. The gum was dissolved in THF (400 mL), DBU (0.40 mL, 2.7 mmol) was added and the reaction was gently refluxed under stirring. After 3 h the solvent was removed in vacuo and the crude was taken up in CH 2 Cl 2 (150 mL), washed with ice/brine (50 mL) and 3% aq. NaHCO 3 (w/v, 2  50mL). The organic layer was dried over anhydrous Na 2 SO 4 , decanted and the solvent was removed in vacuo to produce a yellow gum. Purification was achieved by flash column chromatography using a CH 3 OH:CH 2 Cl 2 solvent system (3 → 4 %, v/v) to afford 1.27 g (88%) of 4a as a colorless foam. R f (SiO 2 TLC): 0.33 CH 3 OH:CH 2 Cl 2 (6 %). λ max(MeCN) 239 nm. 1 Compound 4a (0.59 g, 1.2 mmol) was allowed to stir at 0 o C in a solution of saturated methanolic ammonia (250 mL) and gradually allowed to warm up to room temperature. After 5 h the solvent was removed in vacuo and the resulting residue was washed with Et 2 O (2  25mL). The deprotected nucleoside was partially purified by flash column chromatography (using a short column) with a gradient of CH 3 OH:CH 2 Cl 2 (4 % →15 %, v/v) to elute the product as a colorless powder. This powder was taken up in pyridine (15 mL) and stirred at 0 o C for 15 min. To this solution was added DMAP (cat) and DMT-Cl (0.38 g, 1.1 mmol) in small portions over 20 min under stirring. After 16 h, the solvent was removed in vacuo and the content was taken up in CH 2 Cl 2 (100 mL), washed with 3 % (aq., w/v) NaHCO 3 (2  100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na 2 SO 4 , decanted and the solvent was removed in vacuo to produce a yellow gum. The product was purified via flash column chromatography using a gradient of CH 3 OH:CH 2 Cl 2 (2% → 4%, v/v) to afford 0.32 g (48%) of 5a as a colorless foam.
Compound 4b (0.95 g, 1.9 mmol) was allowed to stir in a solution of saturated methanolic ammonia (500 mL) at 0 o C and gradually allowed to warm to room temperature. After 5 h, the solvent was removed in vacuo and the resulting residue was washed with Et 2 O (3  5 mL). The resulting gum was diluted with pyridine (18 mL) and stirred at 0 o C for 15 min. To this solution was added DMAP (cat) and DMT-Cl (0.38 g, 1.1 mmol) in pyridine (10 mL) dropwise over 25 min under stirring. The reaction was allowed to gradually warm up to room temperature. After 16h the solvent was removed in vacuo and the content was taken up in CH 2 Cl 2 (50 mL) then washed with ice/brine (50 mL) followed by ice/3% (aq.) NaHCO 3 (w/v, 2  50 mL). The organic layer was then dried over anhydrous Na 2 SO 4 , decanted and the solvent was removed in vacuo to produce a yellow-orange gum. The product was purified via flash column chromatography using a gradient of CH 3 OH:CH 2 Cl 2 (3% → 5%, v/v) to afford 0.39 g (36%) of 5b as a colorless foam. R f (SiO 2 TLC): 0.18 CH 3 OH:CH 2 Cl 2 (5%, v/v). λ max(MeCN) 282 nm. 1 To a stirred solution of compound 5a (0.25 g, 0.45 mmol) and DIPEA (0.147 mL, 0.844 mmol) in THF (4.5 mL) was added dropwise Cl-POCENiPr 2 (0.149 mL, 0.674 mmol). After 30 min, the solvent was evaporated in vacuo and the content was diluted with EtOAc (50 mL) then washed with 3% (aq., w/v) NaHCO 3 (2 × 50 mL) and brine (50 mL). The organic layer was dried over anhydrous Na 2 SO 4 , decanted and the solvent was removed in vacuo to produce a yellow gum.
Purification was achieved via short flash column chromatography using EtOAc:hexanes To a stirred solution of compound 5b (0.25 g, 0.44 mmol) and DIPEA (0.143 mL, 0.816 mmol) in THF (4.3 mL) was added dropwise Cl-POCENiPr 2 (0.146 mL, 0.656 mmol). After 30 min the solvent was evaporated in vacuo and the content was diluted in EtOAc (50 mL) then washed with 3 % (aq., w/v) NaHCO 3 (2  50 mL) and brine (50 mL). The organic layer was dried over anhydrous Na 2 SO 4 , decanted and the solvent was removed in vacuo to produce a yellow gum.
Purification was achieved via short flash column chromatography using EtOAc (with 0.

Solid phase synthesis and purification of oligonucleotides.
All DNA sequences were synthesized using an Applied Biosystems Model 3400 synthesizer on a 1.5 μmol scale employing standard β-cyanoethyl phosphoramidite cycles supplied by the manufacturer with modifications to certain coupling times described below. The oligomer-bound CPG support was removed from the column and transferred into screw cap microfuge tubes fitted with Teflon lined caps. The oligonucleotides containing the modifications 3-(2´-deoxypentofuranosyl)-5,6-dihydrofuro[2,3-d]pyrimidin-2(3H)-one (DFP) and 3-(2´deoxypentofuranosyl)-3,5,6,7-tetrahydro-2H-pyrano[2,3-d]pyrimidin-2-one (TPP) were deprotected and cleaved from the solid support with mild deprotection conditions using freshly prepared anhydrous K 2 CO 3 in CH 3 OH (0.05 M) for 3.5 h at room temperature in the dark. The K 2 CO 3 was neutralized with an equimolar amount of acetic acid prior to being transferred into clean vials to separate the solution from the CPG. The CPG was rinsed twice with 250 μL aqueous CH 3 CN (50%, v/v). The crude DNA was dried down in a centrifugal lypophilizer (-Speed vac‖) and then desalted using C-18 SEP PAK cartridges (Waters) prior to purification.
All oligonucleotide sequences were purified from pre-terminated products by strong anion exchange (SAX) HPLC using a Dionex DNAPAC PA-100 column (0.4 cm  25 cm) purchased from Dionex (Sunnyvale, CA) with a linear gradient of 0-52% buffer B, v/v, over 24 min (buffer A: 100 mM Tris HCl, pH 7.5, 10% acetonitrile and buffer B: 100 mM Tris HCl, pH 7.5, 10% S-11 CH 3 CN, 1 M NaCl) at 55 o C. The columns were monitored at 260 nm for analytical runs and/or 280 nm for preparative runs. The purified oligomers were desalted using C-18 SEP PAK cartridges (Waters).

Oligonucleotide characterization by ESI-MS and nuclease digestion.
Mass spectra for the oligonucleotides were acquired at the Concordia University Centre for Biological Applications of Mass Spectrometry (CBAMS) using a Micromass Qtof2 mass spectrometer (Waters) equipped with a nanospray ion source. The mass spectrometer was operated in full scan, negative ion detection mode and the raw data were deconvoluted.
The oligomers (0.05 A 260 units / 0.38 nmol) were characterized by enzymatic digestion (snake venom phosphodiesterase: 0.28 units and calf intestinal phosphatase: 5 units) in buffer consisting of 10 mM Tris HCl, pH 8.1, and 2 mM MgCl 2 for 48 h at 37 o C. The resulting mixture of nucleosides was analyzed by reversed phase HPLC using a Symmetry® C-18 5 µm column (4.6 mm  150 mm, Waters). A linear gradient of 0-70% buffer B (v/v) over 30 min was used to elute the analytes (buffer A: 50 mM sodium phosphate, pH 5.8, 2% CH 3 CN (v/v) and buffer B: 50 mM sodium phosphate, pH 5.8, 50% CH 3 CN (v/v)). The identity of the nucleosides was verified by co-injection with the corresponding standards and eluted at the following times: dC (4.3 min), dG (6.7 min), dT (7.4 min), dA(8.5 min), modified bicyclic pyrimidyl nucleosides (7.7 min for the DFP, and 9.1 min for the TPP nucleosides), and the ratio of unmodified nucleosides was determined. The bicyclic pyrimidyl nucleosides showed poor absorbance at 260nm and improved absorbance at 300 nm. As a result, absorption of the modified nucleosides was monitored at 300 nm and their presence confirmed by an independent injection of the deprotected analogues of 4a and 4b, respectively. The molecular weights of the modified oligomers were determined by ESI-QToF MS and these were in agreement with the calculated values.

UV thermal denaturation studies of DNA duplexes.
Molar extinction coefficients for the modified oligonucleotides were calculated using the nearestneighbour approximations (M -1 cm -1 ) of the mononucleotides and dinucleotides. The molar extinction coefficients for thymidine were used for the modified nucleotides. All duplexes were prepared by mixing equimolar amounts of the interacting strands (0.5 A 260 unit of the strand containing the modification / 3.8 nmol) and lyophilizing the mixture to dryness in a centrifugal concentrator under vaccum. The resulting pellet was dissolved in 90 mM NaCl, 10 mM sodium phosphate, and 1 mM EDTA buffer (pH 7.0) to give a final concentration of 3.8 μM duplex. Before the thermal run, samples were degassed in a centrifugal concentrator for 1 min. Samples were held at 90 o C for 5 min to ensure duplex melting. Annealing profiles were acquired at 260 nm at a rate of cooling of 0.5 o C min -1 , from 90 to 15 o C, using a Varian CARY Model 3E spectrophotometer equipped with a 6-sample thermostated cell block and a temperature controller. Samples were held at 15 o C for 2 min and re-heated at 0.5 o C min -1 to 90 o C showing reversibility (data not shown). The data were analyzed according to the published procedure from Puglisi and Tinoco [6] and transferred to Microsoft Excel TM software.

S-12
Circular dichroism spectra were acquired on a Jasco J-815 spectropolarimeter equipped with a Julaba F25 circulating bath as previously reported. [7] The spectra are an average of 5 scans acquired at a rate of 20 nm min −1 , with a bandwidth of 1 nm and a sampling wavelength of 0.2 nm in fused quartz cells (Starna 29-Q-10). Scans were performed between 350 and 220 nm at 15 °C. The molar ellipticity [θ] was calculated from the equation, θ = ε/Cl, where ε is the relative ellipticity (mdeg), C is the molar concentration of the DNA duplex (M), and l is the path length (cm).

Molecular modeling of DNA duplexes.
Molecular modeling was performed with the Hyperchem 7.5 software package from Hypercube utilizing the AMBER force field. Hybridized oligomers containing a dT·dA, O 4 -MedT·dA, O 4 -EtdT·dA, DFP·dA, and TPP·dA base pair were constructed from the nucleic acid template option using a B-form duplex. Duplexes were solvated with water using a periodic box occupying about 3 times the volume of the duplex alone. Standard Amber99 parameters were used with the dielectric set to constant. -One to four scale factors‖ non-bonded interactions were set to 0.5 (both electrostatic and van der Waals). Cutoffs were applied to -switched‖ to an outer and inner radius of 13.5 and 9.5 Å, respectively. All structures were geometry optimized using Polak-Ribiere conjugate gradient until the RMS gradient was less than 0.1 kcal/(Å mol) using the periodic boundary condition option.

Steady-state kinetics with hPol η.
The 5´-FAM-labeled primer (5´-TCGTAAGCGTCAT-3´) and template DNA (3´-AGCATTCGCAGTAXTACT-5´, where X denotes the modified nucleotide) were mixed in a 1:1 molar ratio and annealed by heating to 90 o C, followed by slow cooling. Steady-state experiments were conducted with typical hPol η concentrations of 1.9-7 nM and 5 µM template-primer duplex substrates in 40 mM Tris HCl buffer (pH 7.5) containing 100 mM KCl, 5mM MgCl 2 , 10 mM DTT, 5% glycerol (w/v), and 100 µg/ml bovine serum albumin (BSA), to which was added varying concentrations of a single dNTP (added last to initiate the reaction). Experiments were carried out at 37 o C and reactions were typically run for 5 to 15 min in order to keep product formation below 20% of the oligonucleotide substrate concentration.
Reactions were terminated with a quench solution containing formamide, EDTA, bromophenol blue and xylene cyanol, and aliquots were applied to an 18% (w/v) acrylamide/7.5 M urea gel and separated by electrophoresis. [8] Fluorescence in the substrate and product primer bands was monitored and quantified using a Typhoon system (GE Healthcare Life Sciences, Pittsburgh, PA) and the data were fit to hyperbolic plots (Michaelis-Menten equation) using the program GraphPad Prism (GraphPad, La Jolla, CA).

LC-MS/MS Analysis of Full-length Extension Products.
The 5´-FAM-labeled primer (5´-TCGTAAGCGUCAT-3´) and template (3´-AGCATTCGCAGTAXTACT-5´ where X denotes the modified nucleotide) were annealed as described above. 2´-Deoxyuridine (U) was included in the primer for facile cleavage of the product to a shorter oligonucleotide (by treatment with uracil DNA glycosylase followed by hot S-13 piperidine) that could be analyzed with an ion-trap mass spectrometer, as previously described. [9,10,11,12,13] DNA primer extension was accomplished by mixing hPol η (76 pmol, 0.95 μM) with template-primer duplex (2 nmol, 10 μM) and a mixture of 1 mM each of dATP, dCTP, dGTP, and dTTP at 37 °C for 1-1.5 h in 50 mM Tris-HCl buffer (pH 7.5), 50 mM NaCl, 5 mM DTT, 5 mM MgCl 2 and 50 µg/mL bovine serum albumin (BSA). The reactions were terminated by spin column separations to extract the dNTPs and Mg 2+ . The extent of the reaction was determined by electrophoresis/fluorography prior to LC-MS analysis. The resulting product was then treated with 25 units of uracil DNA glycosylase and 0.25 M piperidine. [9,10,11,12,13] To identify the products, the resulting reactions were analyzed by LC-MS/MS using an Acquity UPLC system (Waters) interfaced to a Thermo-Finnigan LTQ mass spectrometer (Thermo Scientific, San Jose, CA) equipped with a negative ion electrospray source. Chromatographic separation was achieved with an Acquity UPLC BEH octadecylsilane (C18) column (2.1 mm  100 mm, 1.7 μm). The LC solvent system was as follows: Mobile phase A, 10 mM CH 3 CO 2 NH 4 in 98% H 2 O; mobile phase B, 10 mM CH 3 CO 2 NH 4 in 90% CH 3 CN (v/v). The following gradient (v/v) was used with a flow rate of 300 μL min -1 at a temperature of 50 o C: Linear gradient from 0-3% B (v/v) in 3 min, followed by a linear increase to 20% B (v/v) from 3-5 min, then 20-100% B (v/v) from 5-6 min which is held for 2 min. The column was re-equilibrated for 3 min with 100% A (v/v). MS conditions were as follows: Source voltage, 4 kV; source current 100 μA; capillary voltage,-49 V; capillary temperature, 350 °C; tube lens voltage, -90 V. Product ion spectra were recorded over the range m/z 300-2000 and the most abundant species (-2 charge) was used for collision-induced dissociation (CID) analysis. The calculation for the oligonucleotide sequence CID fragmentation was carried out using the Mongo Oligo Mass Calculator v2.06 from The RNA Institute (College of Arts and Science, University at Albany State University of New York). The relative yields of various products were calculated based on the peak areas of extracted ion chromatograms from LC-MS analyses. The sum of the peak areas was used for multicharged species.

Pre-steady-state experiments
The 5´-FAM-labeled primer (5´-TCGTAAGCGUCAT-3´) and template (3´-AGCATTCGCAGTAXTACT-5´ where X denotes the modified nucleotide) were annealed. Each annealed DNA substrate (1 µM) was mixed with hPol η (500 nM) in 40 mM Tris-HCl buffer (pH 7.5) containing 10 mM DTT, 0.1 mg/ml BSA, 5% glycerol (v/v), and 100 mM KCl (Solution A). Solution B contained 1 mM dATP or dGTP and 10 mM MgCl 2 . The two solutions were mixed rapidly in a KinTek RP-3 instrument (KinTek Corp., Austin, TX) at 37 °C for 0.005 -1 s (the final reaction concentration for hPol η was 250 nM) and stopped by the addition of 0.5 M EDTA. The products were separated with 18% denaturing polyacrylamide gels (w/v) and visualized with a Typhoon system (GE Healthcare). After quantitation using ImageJ software, the results were fit to a burst equation using GraphPad Prism: y = A (1 -e -kpt ) + k ss E o t, where A is the burst amplitude, representing the apparent concentration of the active form of the enzyme, k p is the burst rate, k ss is the steady-state rate, and E o is the total enzyme concentration. [14] Crystallization Crystals were obtained by the hanging drop vapor diffusion technique at 18 °C. O 4 -Alkylthymidine modified DNA solutions were prepared by mixing template and primer strands S-14 in a 1:1 molar ratio and annealing the mixture in the presence of 10 mM sodium HEPES buffer (pH 8.0), 0.1 mM EDTA, and 50 mM NaCl at 85 °C for 5-10 min, followed by slow cooling to room temperature. hPol η protein was mixed with the DNA duplex in a 1:1.2 molar ratio in the presence of 50 mM Tris-HCl, pH 7.5, containing 450 mM KCl, and 3 mM DTT, followed by addition of either 5 µL of 100 mM MgCl 2 or 5 µL of 100 mM CaCl 2 . Using a spin concentrator with Amicon cutoff filter (Millipore, Billerica, MA), the complex was concentrated to a final concentration of ~2 mg/mL. Either dTTP or one of the non-hydrolyzable nucleoside triphosphate analogs was added to the concentrated mixtures containing Ca 2+ or Mg 2+ . The ternary complex solution was mixed with equal volume of reservoir solution containing 0.1 M MES (pH 5.5), 5 mM MgCl 2 , and 16-22% (w/v) PEG 2000 monomethyl ether (MME) and equilibrated against 500 µL reservoir solutions. Crystals typically appeared after overnight incubation and were allowed to grow for one to a few weeks. They were transferred to cryoprotectant solution containing reservoir solution along with 25% glycerol (v/v), and then frozen in liquid nitrogen for data collection.

X-ray Diffraction Data Collection, Structure Determination and Refinement
X-ray diffraction data were collected at 100 K either on the 21-ID-F or the 21-ID-G beamline of the Life Sciences Collaborative Access Team (LS-CAT) at the Advanced Photon Source (APS), Argonne National Laboratory (Argonne, IL). All data were processed with the program HKL2000. 15 Data collection statistics are summarized in Supplementary Table S4. All structures were determined by Molecular Replacement in PHASER, 16,17 using the coordinates of the complex between hPol  and native DNA (PDB ID code 4O3N) 9 as the search model.
Structures were refined either using PHENIX 18 or Refmac 16,19 and model building was carried out in COOT 20 . Model statistics and geometric parameters are summarized in Supplementary  Table S4. Illustrations were generated with the program UCSF Chimera. 21

Synthesis and characterization of nucleosides.
The incorporation of O 4 MedT and O 4 EtdT in DNA oligomers has been described previously. [22] The chemical synthesis for phosphoramidites 6a and 6b is shown in Supplementary Scheme S1. Precursors 1a and 1b were prepared according to published procedures. [1,2,3,4,5] Modified heterocycles 2a and 2b were obtained by a base-catalyzed intramolecular reaction under protic conditions. [2,3] Attachment of heterocycles 2a or 2b to commercially available 1-(α)-chloro-3,5di-O-(p-toluoyl)-2-deoxy-D-ribose (Berry and Associates) by an uncatalyzed Vorbrüggen-type coupling yielded compounds 3a and 3b in moderate yields. [23] Resolution of the mixture of anomers for the latter coupling was challenging. Column chromatography using solvent systems such as CH 3 OH:CH 2 Cl 2 , EtOAc:hexanes, and CH 3 CN:CH 2 Cl 2 with shallow gradients afforded low, if any, resolution of the anomeric mixture. The desired β-anomer was resolved by recrystallization from boiling EtOAc (1:1 EtOAc:hexanes, v/v for 3a and 100% EtOAc for 3b). Resolution of the anomers could also be accomplished by first protecting the free alcohol functionality with a tert-butyldimethylsilyl (TBS) group, followed by column chromatography (EtOAc:hexanes mobile phase) to separate the anomers. Subsequent removal of the TBS group supplied pure 3a and 3b. The bicyclic pyrimidines 4a and 4b were synthesized by first mesylating the terminal hydroxyl group followed by intramolecular cyclization in dilute basic conditions under reflux. A saturated solution of ammonia in methanol removed the toluoyl groups followed by protection of the 5´-hydroxyl group with DMTr to yield mono-protected nucleosides 5a and 5b, which were then phosphitylated with N,N diisopropylaminocyanoethylphosphonamidic chloride in the presence of diisopropylethylamine to produce phosphoramidites 6a and 6b. 31 P NMR signals were observed at 148.27, 148.11 ppm for 6a, and 148.30, 148.03 ppm for 6b, indicating the presence of the non-hydrolyzed phosphoramidite group. NMR and MS data for all small molecules can be found in Supplementary Figures S1 -S32.

Synthesis of DNA containing single modification inserts.
The solid-phase syntheses of DFP and TPP containing oligonucleotide sequences were performed on a 1.5 μmol scale using either phosphoramidite 6a or 6b, respectively. Given the potential lability of the lesions, as seen with other O 4 -alkyl pyrimidine adducts [7] , -fast-deprotecting‖ 3´-O-2´-deoxyphosphoramidites were employed (dissolved to a concentration of 0.1 M in anhydrous acetonitrile) with phenoxyacetic anhydride used as the capping agent to prevent an undesired N-acetylation reaction that could occur with acetic anhydride. [24] Phosphoramidites 6a or 6b were dissolved in acetonitrile to a concentration of 0.12 M with a coupling time of 600 s (as opposed to 120 s) to ensure optimal production of the desired fulllength oligonucleotides.
An ultra-mild oligonucleotide deprotection protocol was required due to the labile nature of the O 4 -alkylpyrimidine adducts. [7] These conditions employed treatment of the support bound oligonucleotide with a 0.05M solution of K 2 CO 3 in anhydrous CH 3 OH for 3.5 h at room temperature in the dark. The resulting crude DNA solution was neutralized with acetic acid to prevent oligonucleotide damage during the evaporation of the solvent in the centrifugal

S-16
concentrator. The resulting pellet was taken up in NaOAc (0.1M), desalted, and reconcentrated. The DNA sequences were purified by SAX-HPLC (see Supplementary Figure S33 for the HPLC chromatographs) and subsequently desalted. ESI mass spectrometry of representative oligonucleotides S DFP and S TPP (with the sequence 5'-GGCTXGATCACCAG-3' where X is DFP and TPP for S DFP and S TPP , respectively) revealed they had molecular weights of 4276.3 and 4290.8 Da (expected 4276.8 and 4289.9 Da), consistent with the expected values (see Supplementary Figures S34 and S35 for the mass spectra). For longer deprotection time under the same conditions, ESI-MS revealed the formation of a product with a mass 32 Da higher than expected. This was most-likely due to the attack of the methoxide nucleophile on the bicyclic pyrimidine moieties during the deprotection step. The composition of S Fur and S Pyr was further confirmed by enzymatic digestion to their constituent nucleosides with a mixture of snake venom phosphodiesterase and calf intestinal phosphatase followed by C 18 RP-HPLC analysis (see Supplementary Figures S36 and S37 for the chromatographs). The presence of the DFP or TPP nucleosides was established by the appearance of one additional peak to the four standard nucleosides, which eluted from the HPLC column with a retention time of 7.7 min for the DFP nucleoside and 9.1 min for TPP nucleoside. The later elution of the TPP relative to the DFP nucleoside can be attributed to the presence of additional methylene linkage in the former, which increases its hydrophobicity. UV scans of DFP and TPP revealed that they had a reduced absorbance at 260 nm (the wavelength at which absorbance is monitored by the HPLC detector to detect the elution of nucleosides from the C 18 column) relative to the four other nucleosides. Thus the C 18 RP-HPLC experiments were simultaneously monitored at both 260 and 300 nm for the modified and standard nucleosides.

UV thermal denaturation of DNA duplexes and mismatch studies.
The influence of the DFP and TPP modifications on duplex stability was assessed by UV thermal denaturation experiments involving oligonucleotides S DFP and S TPP hybridized to complementary DNA (see Supplementary Figure S38). The melting curves of the duplexes containing DFP or TPP were sigmoidal with a comparable hyperchromicity between them that was slightly higher relative to the control duplex. The melting temperatures observed for duplexes containing DFP or TPP with adenine as the base pairing partner on the complementary strand were 48 and 49 o C, respectively, a significant reduction relative to the control duplex (62 o C). Duplexes containing O 4 -methylthymidine or O 4 -ethylthymidine modifications (O 4 MeT and O 4 EtT) were found to have melting temperatures of 51 and 52 o C, respectively, comparable to the destabilizing influence of O 4 -alkylated dT in DNA duplexes that has been reported previously. [25,26] The similar T m values of duplexes containing DFP or TPP versus O 4 MeT or O 4 EtT suggest that the rigidity of the bicyclic pyrimidine system bears little impact on thermal stability relative to adducts with free rotation around the C4-O 4 bond. O 4 -MedT has been shown, through NMR [27] and computational studies [28] , to adopt a wobble base pairing motif when paired with a dA residue. Given the similar trends observed in the T m values, it is reasonable to expect that DFP and TPP can engage in similar hydrogen bond interactions when paired with adenine.
The influence of other base pairings involving the DFP and TPP modifications were also studied by UV thermal denaturation experiments. The T m values of duplexes containing these modifications were equivalent to O 4 MedT or O 4 EtdT when mispaired against cytosine or thymine. An increase in T m was observed for all the O 4 -alkylated thymidine analogs with guanine as the base pairing partner relative to the other pairings. These findings for duplexes containing the bicyclic DFP and TPP modifications are consistent with studies involving O 4 -alkylated dT·dG mispairs. [22,25] Interestingly, previous studies with duplexes containing O 4 MedT and O 4 EtdT modifications reported biphasic thermal profiles with purine mispairs. [25,29] However, this behavior was not observed in this study, perhaps due to differences with the sequence investigated. Cruzeiro-Hansson and Goodfellow's computational analysis showed that the methyl group preferred to adopt the syn conformation for the O 4 -MedT·dG pair. [28] In this orientation, there are three hydrogen bonds between guanine and methylated thymine, namely that of the N 2 H-O 2 (strong), N1H-O 2 (weak), and N1H-N3 (weak), respectively. The bicyclic constructs of DFP and TPP inherently lock the methylene group in the anti conformation, however, hydrogen bonding interactions between the same atoms as the O 4 -MedT·dG pair is possible.

Circular dichroism spectroscopy and molecular modeling of DNA duplexes.
CD spectra of duplexes composed of DFP and TPP paired against adenine on the complementary strand were acquired at 15 o C. The spectra (see Supplementary Figure S39) displayed typical B-form DNA signatures with a wavelength maximum of 280 nm, an intercept at approximately 260 nm, and a minimum around 250 nm, similar to the DNA duplex control (containing a dT·dA base pair). This suggests that the presence of the bicyclic pyrimidine moiety had a minimal influence on the global DNA structure, similar to what has been observed previously in NMR studies of duplexes containing O 4 -MedT. [27] Hybridized oligomers containing a dT·dA, O 4 MedT·dA, O 4 EtdT·dA, DFP·dA or TPP·dA pair were constructed using Hyperchem® 7.5 software package starting from the canonical B-form duplexes. These were subject to geometry optimization using AMBER force field (see Supplementary Figures S40 and S41 for structures of the duplexes with the water molecules omitted for clarity and views of the modified and flanking base pairs from the top). Both optimized structures containing the O 4 MedT·dA and O 4 EtdT·dA base pairs had very similar features with the alpha carbon adopting a syn conformation with respect to the N3-atom, in agreement with published structural data by NMR and results from computational studies. [27,28] The

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Supplementary Figure S51 -Model of the active site configuration in the ternary hPol η insertion-step complex with dGMPNPP opposite TPP (black carbon atoms). The TPP moiety was modeled in place of the ethyl adduct (O 4 EtdT) seen in the crystal structure of the complex. The bicyclic ring protrudes into the major groove of the template-primer duplex, and can be accommodated without clashing with incoming nucleotide triphosphate or amino acid side chains in its vicinity.