Structural Mechanism for the Fidelity Modulation of DNA Polymerase λ. 128 Academia Road Sec. 2, Nankang, Taipei, 115, Taiwan

Transcription

1 SUPPORTING INFORMATION Structural Mechanism for the Fidelity Modulation of DNA Polymerase λ Mu-Sen Liu, 1,3 Hsin-Yue Tsai, 1,# Xiao-Xia Liu, 1,# Meng-Chiao Ho, 1,3 Wen-Jin Wu, 1,* and Ming-Daw Tsai 1,2,3,* 1 Institute of Biological Chemistry and 2 Genomics Research Center, Academia Sinica, 128 Academia Road Sec. 2, Nankang, Taipei, 115, Taiwan 3 Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan *Corresponding author: mdtsai@gate.sinica.edu.tw or winston@gate.sinica.edu.tw SI Materials and Methods SI Tables: Table S1-1 to S1-4 SI Figures: Figure S1 to Figure S7 SI References S1

2 SI Materials and Methods. Materials. [γ- 32 P]ATP was purchased from Perkin-Elmer Life Sciences. dntps were from Sigma-Aldrich. T4 polynucleotide kinase was from Thermo Scientific. KOD hot start DNA polymerase was from Novagen. Oligonucleotides were purchased from Genomics and MDBio, Inc. Crystallization. Crystal of the Pol λ:mnmgdctp binary complex (structures 3a and 3b). The Pol λ:mgdctp binary complex was prepared by mixing purified Pol λ (15 mg/ml) with 5 mm dctp in Buffer A containing 10 mm MgCl 2, 50 mm Tris- HCl, ph 7.5, 200 mm NaCl, 1 mm DTT and 5% glycerol. The crystal of Pol λ:mgdctp binary complex (crystal I) was then obtained by hanging-drop vapor diffusion method at 4 C with the reservoir solution containing 100 mm HEPES (ph 7.5) and 3 M NaCl. However, this crystal diffracted poorly. A well-diffracted crystal of Pol λ:mnmgdctp binary complex (crystal II) was obtained by soaking this crystal I with 1 mm MnCl 2 for 1 h. For data collection, NaCl was increased to 4 M as cryoprotectant. The crystal was found to contain both forms, with and without the 8 kda subdomain (structures 3a and 3b, respectively). Crystal of the Apo-Pol λ (structures 1a and 1b). In an attempt to improve the diffraction of Pol λ:mgdctp crystal, we soaked the crystal I with 1 mm CoCl 2 for 1 h. To our surprise, we found that the soaked crystal (now named crystal III) contained only apo-pol λ (two molecules of Pol λ in one asymmetric unit), which diffracted to a resolution of 3.3 Å. Apparently, after soaking with high concentration of Co 2+ ions, MgdCTP was expelled out of Pol λ. The crystal was found to contain both forms, with and without the 8 kda subdomain (structures 1a and 1b, respectively). Another apo- Pol λ crystal (structure 2a) was unintentionally obtained from the mixture of Pol λ and DNA as described in the next section. Crystals of Pol λ:mgdntp binary complexes (structures 2a, 4a, 5a, and 6a). Crystallization of Pol λ:mgdntp binary complexes involved several steps. Purified Pol λ (15 mg/ml) was first mixed with an annealed unphosphorylated DNA substrate at a molar ratio of 1:1.2 to form a binary complex in Buffer A. The DNA oligonucleotides consist of template (5 -CGGCGGTACTG), upstream primer (5 - GCCG) and downstream primer (5 -CAGTAC) as used by Garcia-Diaz et al. S2

3 previously. 1 The Pol λ:dna complex was crystallized by the hanging-drop vapor diffusion method at 4 C with the reservoir solution containing 100 mm lithium sulfate monohydrate, 100 mm NaHEPES-sodium (ph 7.5), and 100 mm potassium sodium tartrate to obtain crystal IV. 25% ethylene glycol (EG) was added to the crystallization buffer as a cryoprotectant. The crystal structures revealed the coexistence of apo-pol λ (structure 2a, with the 8 kda subdomain lost) and Pol λ:dna binary complex in the same asymmetric unit (ASU). Crystal IV was then soaked with 5 mm datp, dttp and dgtp dissolved in Buffer A for 6 h, which converted the apo-pol λ to the binary complexes Pol λ:mgdatp (structure 4a), Pol λ:mgdttp (structure 5a) and Pol λ:mgdgtp (structure 6a), respectively, with the Pol λ:dna binary complex still remaining in the same ASU. These structures are designated 2a, 4a, 5a, 6a instead of 2, 4, 5, 6 because there is another molecule, Pol λ:dna binary complex, in each crystal. Crystal of the ternary complex containing dg:datp mismatch (structure 7). Purified Pol λ (15 mg/ml) was first mixed with an annealed DNA (the same as above except containing a 5 -phosphate in the downstream primer) at a molar ratio of 1:1.2 to form a DNA binary complex in Buffer B containing 50 mm Tris-HCl, ph 7.5, 200 mm NaCl, 10 mm CaCl 2, 1 mm DTT and 5% glycerol. The Pol λ:dna binary complex was crystallized under the condition of 100 mm HEPES, ph 7.5, and 30% v/v Jeffamine ED-2100 reagent (ph 7.0) by hanging-drop vapor diffusion method at 4 C (crystal V). The dg:datp mismatched ternary complex was obtained by soaking crystal V with 5 mm datp. 25% EG was added as cryoprotectant for data collection. Crystal of the Apo-L431A mutant (structure 8a). The apo-l431a mutant crystal (crystal VI) was obtained in the same way as that for the wild type apo-pol λ (crystal IV, structure 2a). In addition to apo-l431a (structure 8a, with the 8 kda subdomain lost), binary complex of L431A:DNA was also found in the same ASU. Crystals of L431A:MgdNTP binary complexes (structures 9a, 10a, and 11a). The L431A:MgdNTP binary complex crystals were obtained by soaking crystal VI with 5 mm dctp, dttp and dgtp in Buffer A for 6 h, which produced crystals of L431A:MgdCTP (structure 9a), L431A:MgdTTP (structure 10a), and L431A:MgdGTP (structure 11a), respectively, with the L431A:DNA binary complex still remaining in the ASU. In summary, structures 8a, 9a, 10a, and 11a are the L431A S3

4 versions of structures 2a, 4a, 5a, and 6a, respectively, and are also without the 8 kda subdomain. Crystal of the L431A:dG:dCTP ternary complex (structure 12). Purified L431A mutant (15 mg/ml) was first mixed with an annealed DNA (the same as above except containing a 5 -phosphate in the downstream primer) at a molar ratio of 1:1.2 in Buffer B, and then mixed with 5 mm dctp. The L431A:DNA:dCTP complex was crystallized (crystal VII) under the condition of 150 mm KBr, 30% w/v polyethylene glycol monomethyl ether 2000 by the hanging-drop vapor diffusion method at 4 C, with 25% EG in the crystallization buffer as cryoprotectant. Diffraction data collection and structural determination. The crystals were flash-frozen in a stream of nitrogen at 100K. X-ray diffraction data were collected at the 15A1, 13B1 and 13C1 beamlines of National Synchrotron Radiation Research Center (NSRRC) and beamline SP44XU of the SPring-8 and were processed using HKL2000 program suite. 2 The structures were determined by molecular replacement methods using the structure of Pol λ (PDB code: 1XSL) as the search model by the program Phaser-MR from PHENIX. 3 The structures were then iteratively rebuilt in COOT 4 and refined by phenix.refine. Data processing and refinement statistics are summarized in Table S1. Pre-steady-state kinetic measurements. To obtain kinetic parameters (k pol and K d,app ) of dg:datp, dc:datp and all da:dntp incorporations for the L431A mutant, single turnover kinetics was performed. The single nucleotide gapped DNA substrates and the assay conditions were the same as previously described. 5 The buffer contained 5 mm MgCl 2, 50 mm Tri-HCl (ph 8.4), 100 mm NaCl, 0.1 mm EDTA, 5 mm DTT, 10% glycerol and 0.1 mg/ml BSA. The protein sample of L431A mutant (120 nm) was preincubated with 32 P-labeded gapped DNA (30 nm) at 37 C for 3 min, and then mixed with dntp solutions with various concentrations (0.5 to 200 µm). For the reactions with less than 10 s duration, the rapid chemical quench-flow apparatus (KinTek Clarence, PA) was used with 0.37 M EDTA as the quenching solution. Otherwise, the reaction was manually quenched by adding 2 volumes of 92 mm EDTA, 7.5 M urea, and 0.037% bromophenol blue. The quenched reactions were then analyzed on sequencing gel electrophoresis (12% acrylamide, 7 M urea and 1xTBE), S4

5 scanned with Typhoon 9200, and quantified with ImageQuant Version 5.2. After the reaction rate (k obs ) of individual [dntp] was obtained from equation 1 (below), the data was then fit with equation 2 to obtain both k pol and K d,app. Data analysis and curve fitting were performed using Excel (Microsoft Office). Equation 1: [product] = A[1 - exp(-k obs t)] Equation 2: k obs = k pol [dntp]/([dntp] + K d,app ) S5

6 Table S1-1. Data collection and refinement statistics of crystal structures in Table 2. Apo-Pol λ Pol λ:dctp Pol λ:dg:datp ternary (Structure 1a, 1b) (Structure 3a, 3b) (Structure 7) Data Collection a Space group I P P Cell dimension a, b, c (Å) 205.3, 205.3, , 206.0, , 151.3, 83.6 α, β, γ ( ) 90, 90, 90 90, 90, 90 90, 90, 90 Resolution (Å) ( ) ( ) ( ) R merge (%) b 6.9 (51.1) 7.2 (82.5) 14.3 (81.8) I/σI 30.0 (4.5) 26.3 (2.7) 15.7 (3.8) Completeness (%) 99.2 (99.8) 99.8 (99.1) 99.6 (96.6) Redundancy 11.2 (11.5) (6.9) Unique reflections (1758) (3438) (3394) Total reflections Refinement Resolution (Å) R work /R free (%) c 23.0/ / /23.3 Number of protein molecules per asymmetric unit No. atoms Protein DNA/dNTP - -/ /60 Water B-factors (Å 2 ) Protein DNA/dNTP -/- -/ /54.0 Water Metal A/B d -/ e / /61.2 RMS deviations bond length (Å) bond angle ( ) Ramachandran analysis, residues in (% of regions) Most favored Additionally allowed Generously allowed Disallowed PDB ID 5CB1 5DDY 5DKW a Highest resolution shell is shown in parenthesis. b R merge = (Σ Ihkl - <I>)/( ΣIhkl), where Ihkl is the measured intensity for any given reflection, and the average intensity <I> is taken overall symmetry equivalent measurements. c R value = Σ F o - F c /Σ F o, where F o and F c are observed and calculated structure factor amplitudes, respectively. d Metal A/B refers to the metal ions at the A- and B-site, respectively. e The high B-factor could be explained by that the second metal ion (A site, catalytic metal) is dynamic in the absence of DNA. S6

7 Table S1-2. Data collection and refinement statistics of crystal structures in Table 2. Apo-Pol λ Pol λ:datp Pol λ:dttp (Structure 2a) (Structure 4a) (Structure 5a) Data Collection a Space group P P P Cell dimension a, b, c (Å) 83.7, 83.7, , 83.7, , 84.1, α, β, γ ( ) 90, 90, , 90, , 90, 120 Resolution (Å) ( ) ( ) ( ) R merge (%) b 8.2 (49.6) 8.4 (49.1) 15.3 (70.1) I/σI 26.3 (4.2) 24.3 (3.9) 11.3 (2.5) Completeness (%) 99.2 (100.0) (100.0) 99.2 (99.8) Redundancy 8.5 (8.1) 8.2 (7.8) 5.6 (5.4) Unique reflections (2057) (2070) (1705) Total reflections Refinement Resolution (Å) R work /R free (%) c 20.1/ / /25.1 Number of protein molecules per asymmetric unit No. atoms Protein DNA/dNTP 243/- 243/30 243/29 Water B-factors (Å 2 ) Protein DNA/dNTP 26.7/- 30.1/ /41.8 Water Metal A/B d -/- -/31.0 -/35.2 RMS deviations bond length (Å) bond angle ( ) Ramachandran analysis, residues in (% of regions) Most favored Additionally allowed Generously allowed Disallowed PDB ID 5DDM 4XQ8 4XRH a Highest resolution shell is shown in parenthesis. b R merge = (Σ Ihkl - <I>)/( ΣIhkl), where Ihkl is the measured intensity for any given reflection, and the average intensity <I> is taken overall symmetry equivalent measurements. c R value = Σ F o - F c /Σ F o, where F o and F c are observed and calculated structure factor amplitudes, respectively. d Metal A/B refers to the metal ions at the A- and B-site, respectively. S7

8 Table S1-3. Data collection and refinement statistics of crystal structures in Table 2. Pol λ:dgtp Apo-L431A L431A:dTTP (Structure 6a) (Structure 8a) (Structure 10a) Data Collection a Space group P P P Cell dimension a, b, c (Å) 83.1, 83.1, , 83.9, , 84.0, α, β, γ ( ) 90, 90, , 90, , 90, 120 Resolution (Å) ( ) ( ) ( ) R merge (%) b 9.3 (69.4) 6.6 (52.9) 16.5 (86.0) I/σI 19.5 (3.1) 39.4 (5.9) 17.6 (3.9) Completeness (%) 98.3 (94.6) 99.8 (98.6) (100.0) Redundancy 6.7 (6.6) 13.7 (14.2) 14.4 (14.5) Unique reflections (2730) (2814) (1888) Total reflections Refinement Resolution (Å) R work /R free (%) c 20.9/ / /25.8 Number of protein molecules per asymmetric unit No. atoms Protein DNA/dNTP 243/31 243/- 243/29 Water B-factors (Å 2 ) Protein DNA/dNTP 41.2/ /- 30.3/37.2 Water Metal A/B d -/35.5 -/- -/37.1 RMS deviations bond length (Å) bond angle ( ) Ramachandran analysis, residues in (% of regions) Most favored Additionally allowed Generously allowed Disallowed PDB ID 5CA7 5CP2 5CJ7 a Highest resolution shell is shown in parenthesis. b R merge = (Σ Ihkl - <I>)/( ΣIhkl), where Ihkl is the measured intensity for any given reflection, and the average intensity <I> is taken overall symmetry equivalent measurements. c R value = Σ F o - F c /Σ F o, where F o and F c are observed and calculated structure factor amplitudes, respectively. d Metal A/B refers to the metal ions at the A- and B-site, respectively. S8

9 Table S1-4. Data collection and refinement statistics of crystal structures in Table 2. L431A:dGTP L431A:dCTP L431A:dG:dCTP ternary (Structure 11a) (Structure 9a) (Structure 12) Data Collection a Space group P P P Cell dimension a, b, c (Å) 83.9, 83.9, , 84.5, , 148.7, 86.4 α, β, γ ( ) 90, 90, , 90, , 90, 90 Resolution (Å) ( ) ( ) ( ) R merge (%) b 17.0 (87.9) 5.7 (74.6) 9.1 (32.0) I/σI 17.9 (4.0) 34.8 (2.8) 19.4 (3.5) Completeness (%) (100.0) 99.9 (100.0) 98.7 (92.8) Redundancy 13.9 (14.4) 11.4 (10.6) 5.5 (5.2) Unique reflections (1902) (2245) (3872) Total reflections Refinement Resolution (Å) R work /R free (%) c 20.5/ / /23.4 Number of protein molecules per asymmetric unit No. atoms Protein DNA/dNTP 243/31 243/28 854/56 Water B-factors (Å 2 ) Protein DNA/dNTP 30.5/ / /21.2 Water Metal A/B d -/34.5 -/ /27.2 RMS deviations bond length (Å) bond angle ( ) Ramachandran analysis, residues in (% of regions) Most favored Additionally allowed Generously allowed Disallowed PDB ID 5CHG 5CR0 5CWR a Highest resolution shell is shown in parenthesis. b R merge = (Σ Ihkl - <I>)/( ΣIhkl), where Ihkl is the measured intensity for any given reflection, and the average intensity <I> is taken overall symmetry equivalent measurements. c R value = Σ F o - F c /Σ F o, where F o and F c are observed and calculated structure factor amplitudes, respectively. d Metal A/B refers to the metal ions at the A- and B-site, respectively. S9

10 A B C D E F G H I J K L M N Figure S1. Binding curves of MgdNTP to Pol λ measured by ITC. The x-axis is the molar ratio of dntp to protein. (A-D) Binding of WT Pol λ to dgtp, datp, dctp, and dttp, respectively. (E-H) Binding of the L431A mutant to dgtp, datp, dctp, and dttp, respectively. (I-J) Binding of the Y505A mutant to datp and dctp, respectively. (K-N) Binding of the Y505F mutant to dgtp, datp, dctp, and dttp, respectively. The buffer consisted of 50 mm borate/naoh, 150 mm KCl, 2 mm NaN 3, and 4 mm MgCl 2 at ph 6.5. The experiments were performed in triplicates and the average K d MgdNTP values are listed in Table 1. S10

11 Figure S2. Overlay of apo-pol λ structures with the 8 kda lyase subdomain (structure 1a) and without the 8 kda subdomain (structures 1b and 2a) in green, yellow, and orange, respectively. S11

12 Figure S3. Structures of Pol λ:dntp binary complexes. (A) Overlay of the protein structures of all of the Pol λ:dntp binary complexes: Pol λ:mnmgdctp (structures 3a) in yellow, Pol λ:mnmgdctp (structures 3b) in orange, Pol λ:mgdatp (structure 4a) in pink, Pol λ:mgdttp (structure 5a) in green, and Pol λ:mgdgtp (structure 6a) in cyan. For clarity, the bound nucleotide is omitted but is shown separately in (B). (B) Overlay of the bound dntps in all five Pol λ:dntp binary complexes: MnMgdCTP (both structures 3a and 3b), MgdATP, MgdTTP, MgdGTP (structures 4a, 5a, 6a) in yellow, orange, magenta, green and cyan, respectively. S12

13 Figure S4. Structures of apo-l431a and L431A:dNTP binary complexes. (A) The overall structure of apo-l431a (structure 8a in marine) is very similar to that of apo- Pol λ (structure 2a in green) (RMSD = Å), and the YF motif remains in the perpendicular orientation. (B) The global structures of the three L431A:MgdNTP complexes (structure 9a, cyan; structure 10a, light magenta; structure 11a, deep salmon) overlay very well among themselves, and also with that of the WT Pol λ:mnmgdctp binary complex (structure 3a, yellow). For clarity, the bound nucleotide is omitted but is shown separately in (C). (C) The conformations of bound MgdNTP in the L431A:MgdNTP binary complexes (dctp of structure 9a in orange, dttp of structure 10a in marine, and dgtp of structure 11a in magenta) are nearly unchanged from that in the WT Pol λ:mnmgdctp binary complex (dctp of structure 3a in yellow). S13

14 Figure S5. Conformations of the bound dntp and the YF motif in the L431A:dNTP binary complexes. (A) The electron density maps of the YF motif and bound dntp in the L431A:dNTP binary complexes (structures 9a, 10a and 11a, respectively). The final 2Fo-Fc map (contoured at 1σ) shows a tendency that Phe506 can rotate to a parallel position in a and b. However, C ε and C ζ atoms of Phe506 in a and b lack clear densities. (B) Relative B-factors in the YF motif and bound dntp. The rainbow color bars show that the B-factors range from low (blue), medium (green) to high (red color). S14

15 Figure S6. The final 2Fo-Fc map (contoured at 1σ) showing the Ca 2+ ion, water molecule, templating dg, and bound datp in the mismatched ternary complex of Pol λ with dg:cadatp (structure 7). S15

16 Figure S7. The final 2Fo-Fc map (contoured at 1σ) showing the YF motif, templating dg, and bound dctp in conformer A (red) and conformer B (green) in the dg:dctp matched ternary complex of L431A (structure 12). S16

17 SI References (1) Garcia-Diaz, M.; Murray, M. S.; Kunkel, T. A.; Chou, K.-m. J. Biol. Chem. 2010, 285, (2) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307. (3) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. Acta Crystallogr D Biol Crystallogr 2010, 66, 213. (4) Emsley, P.; Cowtan, K. Acta Crystallogr D Biol Crystallogr 2004, 60, (5) Fiala, K. A.; Abdel-Gawad, W.; Suo, Z. Biochemistry 2004, 43, S17