Methods and Assays for Chapter II

2.5.1 Site-directed Mutagenesis of Y671A, Y671F and Y671W

Genes encoding for KlenTaq mutants Y671A and Y671W were constructed by Dr. Ramon Kranaster (Dissertation, 2010, Universität Konstanz).

To introduce the Y671F mutation in KlenTaq wild-type gene encoded on the pGDR11 vector,^[184] whole plasmid PCR was performed according to the QuickChange® Site-Directed Mutagenesis Kit Protocol by Stratagene, described in chapter VI 2.1.3. For PCR, Phusion DNA polymerase and supplied GC-buffer were used in combination with the following primers P-Y671F-2 fwd and P-P-Y671F-2 rev. Transformation was conducted in E. coli XL10. Sequencing of the open-reading frame was performed by GATC Biotech (Germany) using the following primers: pQE-FP, pQE-RP and pQE-KTQ-mid. Purification of the mutant enzymes followed the protocol described in chapter VI 2.4.1.1. Purification of KlenTaq wild-type, employed for studies described in chapter II, was performed by Dr. Christian Glöckner (Dissertation, 2008, Universität Konstanz) in preliminary work.

performed by phosphoimaging. Blunt-end extension experiments were conducted as described above using DNA primer (P-BluntEnd) and template (T-BluntEnd) that form a blunt-end only at one terminus.

2.5.3 Primer Extension Experiments with KlenTaq F

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Incorporation opposite F with KlenTaq wild-type and F3Y and all four nucleotides, respectively: Extension experiments were conducted as described in chapter VI 2.5.2 using 100 nM primer P-F23, 130 nM F-containing template F33XA, 100 µM of the respective dNTP and 25 nM of KlenTaq DNA polymerase in 1x RQF Reaction Buffer. Incubation times are provided in the respective figure legends.

Incorporation opposite F or dT with a polymerase dilution series: Experiments were performed as described in chapter VI 2.5.2 using 100 nM primer P-F23, 130 nM F-containing template F33XA or the respective natural DNA template F33-7T, 100 µM of the respective dNTP as well as the respective polymerase concentration (50, 25, 10, 5, 2.5, 1 nM) in 1x RQF Reaction Buffer. Reaction mixtures were incubated at 37 °C for 5 s in case of the natural template and 30 min for the F-containing template.

Incorporation opposite F in a time-dependent manner: Experiments were performed as described in chapter VI 2.5.2 using 100 nM primer P-F23, 130 nM F-containing template F33XA, 100 µM of the respective dNTP and 1 µM polymerase of the respective KlenTaq DNA polymerase in 1x RQF Reaction Buffer. Incubation times are provided in the figure legends.

2.5.4 Primer Extension Experiments with Natural Abasic Site

Reaction conditions were employed as described in chapter VI 2.5.2. 20 µL of the reactions contained 100 nM primer P-F23, 130 nM F-containing template F33XA or dU-containing template F33dU, respectively, 100 µM dNTPs in 1x RQF Reaction Buffer, 0.16 µL UDG (5*10^-4 u/µL) and 500 nM of the respective KlenTaq DNA polymerase. Reaction mixtures were annealed at 95 °C for 2 min without UDG, KlenTaq DNA polymerase and dNTPs present. After

cooling to 0 °C, UDG was added and the reaction mixture incubated for 18 h at 37 °C. Next, KlenTaq DNA polymerase was added on ice and the reaction started by addition of dNTPs at 37 °C (reaction temperature). Incubation times are provided in the respective figure legends.

Primer was labelled using [³²P]-ATP as described in chapter VI 2.2.1. Reactions were stopped by addition of 45 µL 1x Denaturing PAGE Loading Dye, denatured at 95 °C for 5 min and analysed by 12 % denaturing PAGE. Visualization was performed by phosphoimaging.

To check for conversion of dU to a natural abasic site, both templates were labelled with [

-32P]-ATP and submitted to the same conditions described above. After incubation with UDG for 18 h at 37 °C, 1x Denaturing PAGE Loading Dye and NaOH were added. Heat-denaturation for 10 min at 95 °C in presence of NaOH should cleave a natural abasic site, but not the abasic site analogue F. Conversion was analysed by 12 % denaturing PAGE. Visualization was performed by phosphoimaging.

2.5.5 Pre-steady State Kinetic Analysis

The rate of single turnover in single nucleotide incorporation experiments was determined by rapid quench flow kinetics using a chemical quench flow apparatus (RQF-3, KinTek Corp., University Park, PA) as described earlier (Dr. Christian Glöckner, Dissertation, 2008, Universität Konstanz). In short, 15 µL of an enzyme/primer/template mix and 15 µL dNTP solutions were filled manually in two sample loops. A three-way valve was used to put the loaded sample loops in line with syringes containing the reaction buffer (1x RQF Reaction Buffer). A drive motor was used to apply pressure to the syringes which pushed the reaction buffer into the sample loops. Thus, both solutions were mixed in a first mixing chamber and passed into a reaction delay line where the reaction occured. Reaction times from 2 ms to 100 ms were varied by the length (volume) of the reaction delay line and the flow rate through the reaction delay line. For longer reaction times, the drive motor paused after the first push to mix the reactants together and then after a specific time period performed a second push which added the quenching solution to the reaction in a second mixing chamber.

After termination of the reaction by adding the quenching solution, the sample was collected and product formation was determined by denaturing PAGE. For reaction times longer than 5 s, a manual quench was performed.

In detail, 15 µL of radioactively labelled primer P-F23 (200 nM), annealed to the F-containing template F33XA (260 nM) or natural DNA template F33-7T (260 nM), and KlenTaq DNA polymerase (2 µM) in 1x RQF Reaction Buffer were rapidly mixed with 15 µL of a dNTP solution in buffer at 37 °C. Quenching was achieved by adding 0.3 M EDTA solution at defined time intervals before mixing with 1x Denaturing PAGE Loading Dye. Due to the fact that the volume of the quenched samples collected from the quench flow apparatus after different time points varied, different volumes of 0.3 M EDTA were added to adapt all samples to one total volume. Next, quenched samples were analysed by 12 % denaturing PAGE. Visualization

2.5.6 MALDI/MS-MS Analysis of KlenTaq F

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The expressed protein KlenTaq F3Y was in-gel digested with trypsin and the resulting fragments were analyzed by MALDI-MS/MS at the ‘Functional Genomics Center Zurich’.