Mutation sites in KOD pol.

<p>(A) Crystal structure of a binary complex of KOD pol (PDB code 4K8Z [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183623#pone.0183623.ref048" target="_blank">48</a>]. The sections shown in detail, illustrate how the investigated amino acids interact with the primer or template backbone. (B) Schematic drawing of the primer-template duplex. Amino acids and their respective interactions with the DNA substrate are indicated by arrows according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183623#pone.0183623.ref048" target="_blank">48</a>].</p

Variants of sequence family B <i>Thermococcus kodakaraensis</i> DNA polymerase with increased mismatch extension selectivity

<div><p>Fidelity and selectivity of DNA polymerases are critical determinants for the biology of life, as well as important tools for biotechnological applications. DNA polymerases catalyze the formation of DNA strands by adding deoxynucleotides to a primer, which is complementarily bound to a template. To ensure the integrity of the genome, DNA polymerases select the correct nucleotide and further extend the nascent DNA strand. Thus, DNA polymerase fidelity is pivotal for ensuring that cells can replicate their genome with minimal error. DNA polymerases are, however, further optimized for more specific biotechnological or diagnostic applications. Here we report on the semi-rational design of mutant libraries derived by saturation mutagenesis at single sites of a 3’-5’-exonuclease deficient variant of <i>Thermococcus kodakaraensis</i> DNA polymerase (KOD pol) and the discovery for variants with enhanced mismatch extension selectivity by screening. Sites of potential interest for saturation mutagenesis were selected by their proximity to primer or template strands. The resulting libraries were screened via quantitative real-time PCR. We identified three variants with single amino acid exchanges—R501C, R606Q, and R606W—which exhibited increased mismatch extension selectivity. These variants were further characterized towards their potential in mismatch discrimination. Additionally, the identified enzymes were also able to differentiate between cytosine and 5-methylcytosine. Our results demonstrate the potential in characterizing and developing DNA polymerases for specific PCR based applications in DNA biotechnology and diagnostics.</p></div

Real time PCR of prothrombin factor II Human gDNA with KOD pol wildtype and mutants.

<p>Real time PCR curves when using a forward primer that terminates opposite the SNP site in either a matched or mismatched fashion, depending on the template. (A) real time PCR curves obtained when using KOD pol wildtype. No discrimination is seen for either of the DNA samples: G-wt (genotype G/G), A-heterozygous (genotype G/A) or A-homozygous (genotype A/A). (B)+(C)+(D) real time PCR curves when using KOD pols R501C, R606Q, and R606W, respectively. Discrimination is seen for the amplification of the A-homozygous (A/A) Factor II template which is severely slowed down when using the KOD mutants. (E) agarose gel electrophoresis analyzing PCR products from (B)-(D). M = marker. PCR products are only visible in samples containing template DNA with at least one allele that results in a matched primer-template duplex (C-primer with G/G and G/A DNA template); the homozygous A/A template does not yield a PCR product.</p

Real time PCR of a synthetic prothrombin Factor II oligonucleotide with KOD wildtype and mutants.

<p>(A) primer and (partial) template sequence from the prothrombin Factor II gene. The 3’ end of the primer was either matched or mismatched to the template. (B) real time PCR curves obtained when using KOD pol wildtype. The enzyme distinguishes only little when amplifying from matched (C-G) or mismatched (C-A) primer-template duplex. (C) + (D) + (E) real time PCR curves obtained by using KOD R501C, KOD R606Q and KOD R606W respectively. Increased discrimination is shown, as indicated by slower PCR amplification from mismatched (C-A) primer-template duplexes. R501C barely reaches saturation after 30 cycles; both R606 variants are almost inactive in the mismatch case under the applied conditions.</p

Single nucleotide primer extension experiments using KOD pol wildtype and mutants.

<p>(A) primer and (partial) template sequence from the prothrombin factor II gene. The 3’ end of the radioactively labeled primer was either matched or mismatched to the template. (B) PAGE analysis of single nucleotide extension of the primer by either KOD pol wildtype or mutants, as indicated. Reactions were carried out for the indicated times. (C) + (D) percentage of primer conversion vs time for match and mismatch case, respectively. Gel bands were quantified with the Quantity One software from BioRad, results are shown as means +/- SD of three independent experiments. Graphs were prepared with Origin2015.</p

Real time PCR for cytosine methylation detection.

<p>(A) The chosen forward primer terminates opposite either C or 5mC, depending on the template. Two forward primers were used, carrying either G or A at their 3’ end, resulting in matched or mismatched primer-template duplex respectively. (B) real time PCR curves using KOD pol wildtype. Equal amplification is observed for all four primer/template samples. (C) real time PCR curves using KOD pol R501C. Amplification from the matched primer is not distinguishable between C or 5mC. Amplification from the mismatched primer is in both cases slightly slower, but not significantly different between C and 5mC. (D) + (E) real time PCR curves using KOD pol R606Q and KOD pol R606W, respectively. Matched primer-template duplexes are amplified without discrimination and comparable to KOD pol wildtype. In the mismatch case, methylated DNA is clearly favored, demonstrating the ability of the enzyme variants to discriminate between C and 5mC.</p

Analysis of steady-state kinetics of dGMP incorporation after either matched or mismatched primer-template substrate.

<p>Analysis of steady-state kinetics of dGMP incorporation after either matched or mismatched primer-template substrate.</p

Results of screening the KOD pol library as activity chart.

<p>Amino acids of interest are given on the left, amino acid exchanges are depicted at the top in single letter code. Colors indicate activity of mutant proteins measured as Ct values in real time PCR screening assays using bacterial lysates. Darker shades of blue indicate lower Ct values, grey shows PCR inactive mutants. Boxes marked with asterisks show variants that could not be obtained during several rounds of cloning.</p

Synthesis and Stability of Phosphate Modified ATP Analogues

Nucleotides modified at the phosphate have numerous applications. Nevertheless, the number of attachment modes is limited and little is known about their stability. Here, we present results on the elaboration of the synthesis of five classes of ATP analogues and studies concerning their stability. We show that the nitrogen-linked ATP analogue is less stable, whereas the oxygen- and novel carbon-linked adenosine tri- and tetraphosphate analogues are stable from pH 3 to 12 rendering them interesting for further applications and designs

Direct Monitoring of Nucleotide Turnover in Human Cell Extracts and Cells by Fluorogenic ATP Analogs

Nucleotides containing adenosine play pivotal roles in every living cell. Adenosine triphosphate (ATP), for example, is the universal energy currency, and ATP-consuming processes also contribute to posttranslational protein modifications. Nevertheless, detecting the turnover of adenosine nucleotides in the complex setting of a cell remains challenging. Here, we demonstrate the use of fluorogenic analogs of ATP and adenosine tetraphosphate to study nucleotide hydrolysis in lysates of human cell lines and in intact human cells. We found that the adenosine triphosphate analog is completely stable in lysates of human cell lines, whereas the adenosine tetraphosphate analog is rapidly turned over. The observed activity in human cell lysates can be assigned to a single enzyme, namely, the human diadenosine tetraphosphate hydrolase NudT2. Since NudT2 has been shown to be a prognostic factor for breast cancer, the adenosine tetraphosphate analog might contribute to a better understanding of its involvement in cancerogenesis and allow the straightforward screening for inhibitors. Studying hydrolysis of the analogs in intact cells, we found that electroporation is a suitable method to deliver nucleotide analogs into the cytoplasm and show that high FRET efficiencies can be detected directly after internalization. Time-dependent experiments reveal that adenosine triphosphate and tetraphosphate analogs are both processed in the cellular environment. This study demonstrates that these nucleotide analogs indeed bear the potential to be powerful tools for the exploration of nucleotide turnover in the context of whole cells