Supporting Information

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1 Supporting Information Wiley-VCH Weinheim, Germany

2 Evolving a Thermostable DNA polymerase that Accurately Amplifies Highly-Damaged Templates Christian Gloeckner, Katharina B. M. Sauter, and Andreas Marx* Department of Chemistry, University of Konstanz, Konstanz, Germany. Andreas.Marx@uni-konstanz.de Screening Klentaq variants with altered properties Klentaq mutants were expressed as described before by Sauter et al. (K.B.M. Sauter, A. Marx, Angew. Chem. Int. Ed. 2006, 45, ). Briefly, 1 ml cultures were grown in 96 deep well plates, induced by anhydrotetracycline and lysed after expression. Host proteins were heat denatured and removed by centrifugation. The resulting lysates were directly used for screening. Reaction mixtures of 15 µl contained 150 nm template, 225 nm primer, 1x KTQ reaction buffer (50 mm Tris-HCl ph 9.2, 16 mm (NH 4 ) 2 SO 4, 2.5 mm MgCl 2, 0.1% Tween 20), and 200 µm of dgtp, dctp and dttp and 5 µl lysate, which was added by using an automated liquid handling device (Hamilton Microlab Star). Positive controls contained all four dntps, each at a concentration of 200 µm and 5 µl lysate solution of Klentaq wt. Additional controls were achieved by incubating Klentaq wt lysates with reaction mix excluding dp. 384 well plates were sealed and incubated at 72 C for 15 min. Reactions were quenched by addition of 30 µl SYBR-stop solution (50 mm Tris-HCl ph 9.2, 10 mm EDTA) containing 3.4 x SYBRgreen I (Molecular probes) for quantification of dsdna generated by Klentaq variants. Fluorescence intensities were quantified using a fluorescence plate reader (Polarstar Optima, BMG Labtechnologies GmbH) with excitation at 485 nm and emission at 520 nm. Reactions comprised 20mer primer F20H (5 -(CGT TGG TCC TGA AGG AGG )-3 ) and 90mer template F90A (5 -d(ccg TCA T GTG CCG TCG C A ACG C C CGT GGA CAG AGG ACT A GAA A CAA CCT C CTC CTT CAG GAC CAA CGT ACA GAG)-3'). For identifying a hit from the Klentaq library that was able to extend the F20H primer in presence of only three dntps a threshold was defined as follows. The fluorescence intensity threshold (F t ) was calculated by subtracting the fluorescence intensity from the WT incubated with three dntps (F wt/3dntps ) from the fluorescence intensity of the WT incubated with all four dntps (F wt/4dntps ). Half of this fluorescence difference ( F) was then added to F wt/3dntps giving the fluorescence intensity threshold (F t = F wt/3dntps + F/2). Mutant enzymes that gave rise to fluorescence values equal or above the F t value were regarded as a hit. Those variants were further characterized in a radiometric primer extension assay. Expression and purification of Klentaq WT and mutant M747K Klentaq wt and mutant M747K were purified in batch technique using Ni-NTA sepharose from Amersham Biosciences. In detail 100 ml cultures were grown to an OD 600 of 1.0 at 37 C following induction with AHT (200µg/L) for 1 h. After harvesting cells were lysed in 5 ml lysis buffer (300 mm NaCl, 2,5 mm MgCl 2, 10 mm TrisHCl ph 9.2, 0.1% Triton X 100, 1 mm benzamidin, 1 mm PefaBlock, 1 mg/ml lysozyme) followed by heat denaturation of host proteins at 72 C and centrifugation at x g for 30 min. Supernatants were incubated with Ni-NTA sepharose slurry. After washing elution was carried out two times using elution buffer ( 2x KTQ buffer w/o (NH 4 ) 2 SO 4 : 100 mm TrisHCl ph 9.2, 5 mm MgCl 2, 0.2 % Tween 20, 200 mm imidazol). Glycerol was added to 50 % and (NH 4 ) 2 SO 4 to a final concentration of 16 mm. Protein concentrations were determined using Bradford assay. Purified enzymes were stored at -20 C. Figure S1: SDS-PAGE of purified Klentaq DNA polymerases (coomassie staining). 1

3 Primer extension assays 20 µl of the reaction contained 160 nm DNA polymerase, 150 nm primer F20H (5 -(CGT TGG TCC TGA AGG AGG )-3, 225 nm template, 100 µm dntps in 1x KTQ reaction buffer. Used template sequences were i) for reactions depicted in Figure 1: 5 -d(gac T AGA AAA TCA ACC T CCT CCT TCA GGA CCA ACG TAC)- 3; ii) reaction depicted in Figure 2: F33A (5 -d(aaa TCA ACC T CCT CCT TCA GGA CCA ACG TAC)-3 ), F33X (5 -d(aaa TCA XCC T CCT CCT TCA GGA CCA ACG TAC)-3 ), where X stands for abasic site analogue, F33oxoA (5 - d(aaa TA*A ACC T CCT CCT TCA GGA CCA ACG TAC)- 3), where A* stands for 8-oxo-dA and F33oxoG (5 - d(aaa TG*A ACC T CCT CCT TCA GGA CCA ACG TAC)- 3), where G* stands for 8-oxo-dG. F20H was labelled using [γ 32 P]-P according to standard techniques. Reactions were incubated for 1h at 72 C in a thermal cycler and were stopped by addition of 40 µl stop solution (80 % [v/v] formamide, 20 mm EDTA, % [w/v] bromophenol blue, % [w/v] xylene cyanol). Reactions were separated using a 12 % denaturing PAGE. Visualization was performed using phosphoimaging. DNA Polymerase activity determination For activity determination primerextension reactions were carried out with varying concentrations of DNA polymerase. 20 µl reactions contained 150 nm primer F20H, 225 nm template F33A, 100 µm dntps in 1xKTQ reaction buffer. Reactions were performed at 72 C for 10 min, subsequently quenched with stop solution and products were separated on a 12 % denaturing PAGE. Phosphoimaging was carried out and the obtained intensities for each band were transformed into dntp conversion. Enzyme concentration was plotted against dntp conversion and the resulting slopes were compared. Table S1: Specific activities of Klentaq DNA polymerases [fmol dntp incorporation/fmol pol] wild-type M747K specific activity Error spectrum The error spectrum for both polymerases was determined using a PCR based assay described by Loeb et al. (P. H. Patel, H. Kawate, E. Adman, M. Ashbach, L. A. Loeb, J. Biol. Chem. 2001, 276, ). In short, a ~1.8 kb fragment encoding for Klentaq wt was amplified followed by subsequent cloning of the PCR product and sequencing. PCR reactions contained 31.5 fmol plasmid harboring the target gene, 400 nm each primer, 100 µm dntps, 80 nm DNA polymerase in 1 x KTQ buffer. 30 PCR cycles were performed with 60 s at 94 C, 60 s at 65 C and 120 s at 72 C. Products were purified using agarose gelelectropheresis and cloned into pask-iba37plus (IBA) vector. The cloned vector was transformed into E.coli and single colonies were randomly picked and a stretch of 600 bp was sequenced for each isolated plasmid. The mutation frequency equals number of mutations per clone and per bp sequenced (600 bp). The error rate was calculated as mutation frequency per number of replications. Replications were calculated from the PCR yield determined by agarose gelelectrophoresis. Replications were 7.7 for Klentaq WT (amplification of ~ 210-fold) and 7.5 for mutant M747K (amplification of ~180-fold). Table S2: Error rate and error spectrum following PCR Enzyme No. of clones Average no. of mutations per clone a b Error rate No. of individual substitutions Transitions TA Transversions CG TA CG No. of deletions Klentaq wt ,8 x Klentaq M747K ,5 x a Number of mutations per 600 bases sequenced per clone. b Error rate equals number of mutations per base per division. No. of insertions Steady-state kinetic analysis All kinetic reactions were performed under single completed hit conditions. [1,2] Reaction mix contained 300 nm primer and 600 nm template, 2 x KTQ buffer and DNA polymerase. 10 µl reaction mix were incubated at 55 C, 2

4 reaction was started by addition of 10 µl prewarmed dntps with varying concentrations and stopped using 40 µl stop solution (see above). Template F33+T (5 -AAA TCA ACC T CCT CCT TCA GGA CCA ACG TAC-3 ) and primer F20H were used to measure incorporation of dp, dgtp, dctp and dttp opposite to dt, Template F33+T and primers F21A, F21G, F21C, F21T (5 CGT TGG TCC TGA AGG AGG A/G/C/T-3 ) ending with either da, dg, dc or dt were used to determine mismatch extension opposite template dt by dgtp incorporation. Template F33X was used with primer F23 (5 -CGT TGG TCC TGA AGG AGG A GG-3 ) to measure incorporation of dntps opposite to the abasic site, primer F24A and F24G (5 -CGT TGG TCC TGA AGG AGG A GGA/G-3 ) to measure the extension of either da or dg opposite to the abasic site. Reactions were separated using a 12 % denaturing PAGE and analyzed as described. [1,2] Table S3: Insertion and Misinsertion Enzyme dntp k cat k cat / Relative efficiency a [min -1 ] [µm] [M -1 min -1 x 10 3 ] KTQ WT A G x 10-4 C x 10-4 T x 10-4 KTQ M747K A G x 10-4 C x 10-4 T x 10-4 a Relative efficiency equals efficiency (k cat / ) relative to that of dp incorporation. Table S4: Mismatch Extension efficiency Enzyme P / T dn /dx dntp k cat [min -1 ] [µm] k cat / [M -1 min -1 x 10 3 ] Relative efficiency a KTQ WT da / dt G dg / dt G x 10-4 dc / dt G x 10-4 dt / dt G x 10-4 KTQ M747K da / dt G dg / dt G x 10-4 dc / dt G x 10-4 dt / dt G x 10-4 a Relative efficiency equals efficiency (k cat / ) relative to that of da:dt extension. Table S5: Insertion efficiency opposite to abasic site analogue Enzyme dntp k cat k cat / Relative efficiency a [min -1 ] [µm] [M -1 min -1 x 10 3 ] KTQ WT A KTQ M747K A KTQ WT G KTQ M747K G KTQ WT C n.a. n.a. n.a. n.a. KTQ M747K C n.a. n.a. n.a. n.a. KTQ WT T n.a. n.a. n.a. n.a. KTQ M747K T n.a. a Relative efficiency equals efficiency (k cat / ) relative to that of WT incorporation. n.a.: not accessible. 3

5 Table S6: Extension efficiency from abasic site analogue Enzyme P / T dn /dx dntp k cat [min -1 ] [µm] k cat / [M -1 min -1 x 10 3 ] Relative efficiency a KTQ WT da / dx T KTQ M747K da / dx T KTQ WT dg / dx T n.a. n.a. n.a. n.a. KTQ M747K dg / dx T n.a. n.a. n.a. n.a. a Relative efficiency equals efficiency (k cat / ) relative to that of WT incorporation. X: abasic site analogue. n.a.: not accessible since no elongation could be observed. UV-irradiation of plasmid DNA and PCR amplification of damaged DNA The outlay of this experiment is depicted in Scheme S Scheme S1: UV-irradiation of double-stranded plasmid DNA (1.) and subsequent PCR amplification of an 1.8 kb DNA fragment (2.) using the irradiated DNA as template. 100 µl aliquots of an aqueous plasmid solution with a concentration of 100 ng/µl were irradiated at λ = 254 nm using a Stratalinker UV Crosslinker 1800 (Stratagene). The used plasmid was pask-iba37plus KTQwt. Samples were placed on ice into the irradiation device and incubated for varying times to reach the indicated energy levels. Subsequent analysis of plasmid DNA by agarose gel electrophoreses showed the impact of increasing exposure to UV light onto the plasmid. A 1.8 kb DNA fragment (Klentaq ORF) encoded in the UV damaged plasmids was amplified using sequencing primers for pask37+iba vector (Seq-primer fw 5 -d(gagtttttaccactccct)-3, Seq-primer rev 5 - d(cagtaggtaaacg)-3. The resulting PCR product was expected to have a length of bp. 20 µl reactions contained 2.5 fmol template, 200 nm primers, 200 µm dntps, 80 nm DNA polymerase in 1x KTQ buffer. After an initial denaturation at 94 C for 3 min, 30 cycles with 94 C for 1 min, 59 C for 1 min and 72 C for 4 min were run, followed by 72 C for 10 min. PCR products were analyzed on a 0.8 % agarose gel in 0.5 x TBE buffer. Sequencing of PCR products from UV damaged templates We amplified a template that resulted from irradiation of the plasmid with a dose of 50 kj/m 2. After cycling PCR reactions were cleaned up from template and primers using agarose gelelectrophoresis. The DNA band was excised from the gel and purified using a Min Elute Kit (Qiagen). The prepared samples were sent to a professional sequencing company (Eurofins Medigenomix GmbH, Munich, Germany). 4

6 Figure S2: Section of the electropherogram of the PCR product amplified by variant M747K on UV damaged template with 50 kj/m 2. KTQ_ORF_rc TATCCTGG GAGAGGGT GGCGAC CGTAGAGG ACCCCGAAGT M747K_50kJ T CGTAGAGG -CCCCGAAGT KTQ_ORF_rc TGGGTCTT GCCCGG CCAGGG GGTCCACG CTCCCGGGGG M747K_50kJ TGGGTCTT GCCCGG CCAGGG GGTCCACG CTCCCGGGGG KTQ_ORF_rc ACCGAACA TCCATG GGTCTCCGTG TGGGTCCC CCCTCCTG M747K_50kJ ACCGAACA TCCATG GGTCTCCGTG TGGGTCCC CCCTCCTG KTQ_ORF_rc GAAGACCCGG CAGGTTCT CGTCCGGA GAGGTGGC AACCCTGA M747K_50kJ GAAGACCCGG CAGGTTCT CGTCCGGA GAGGTGGC AACCCTGA KTQ_ORF_rc TCTCTG TAGTCC AGGCACCA ACACCC CTCCTCGG M747K_50kJ TCTCTG TAGTCC AGGCACCA ACACCC CTCCTCGG KTQ_ORF_rc GAAGCC GGCT CTCCAA GGGGTGGA CGGGGGTT M747K_50kJ GAAGCC GGCT CTCCAA GGGGTGGA CGGGGGTT KTQ_ORF_rc CTGGAGGTTG GGGA TACTTACT CCGTGC GTGCGTCT M747K_50kJ CTGGAGGTTG GGGA TACTTACT CCGTGC GTGCGTCT KTQ_ORF_rc GGTTGAAG GGTGTGGAGG CGCCGTCC TGGGGTGG GAGGTCCG M747K_50kJ GGTTGAAG GGTGTGGAGG CGCCGTCC TGGGGTGG GAGGTCCG KTQ_ORF_rc AAGGGGTCAA TGTAGGTT CTTCATTG GTGATCCC GGTACTAG M747K_50kJ AAGGGGTCAA TGTAGGTT CTTCATTG GTGATCCC GGTACTAG KTQ_ORF_rc TTCTCC ACGGGGGT GGCTCG GAGGCTCC AGGACGGG M747K_50kJ TTCTCC ACGGGGGT GGCTCG GAGGCTCC AGGACGGG KTQ_ORF_rc CTGGTGGA TTCG GTCTTCTCCG TCTTCG GGGGAA M747K_50kJ CTGGTGGA TTCG GTCTTCTCCG TCTTCG GGGGAA KTQ_ORF_rc CCTATCGT CAAAGAGGAC CCTTTCCA TGGTCCCGGG AGTTGAGGTT M747K_50kJ CCTATCGT CAAAGAGGAC CCTTTCCA TGGTCCCGGG AGTTGAGGTT KTQ_ORF_rc GAAGGGGTGG CCGCAG GGAAGACCTC GCTCGAGG CGGT M747K_50kJ GAAGGGGTGG CCGCAG GGAAGACCTC GCTCGAGG CGGT KTQ_ORF_rc CCTCGCAC CTCCAGGGAC AAGCCTGA GAGCAC GTCCAG M747K_50kJ CCTCGCAC CTCCAGGGAC AAGCCTGA G Figure S3: Sequence alignment of the reverse complement sequence of the Klentaq WT ORF with the sequenced PCR product derived from an irradiated DNA template (50 kj/m 2 UV light). 1. Boosalis, M. S., Petruska, J., and Goodman, M. F. (1987) J Biol Chem 262, Creighton, S., Bloom, L. B., and Goodman, M. F. (1995) Methods Enzymol 262,