2.5.1 Introduction
Replicative DNA polymerases are usually synthesising DNA in a template dependent manner according to Watson and Crick base pairing. Two exceptions are described for the unusual case of non-templated DNA synthesis by replicative DNA polymerases:
Abasic site bypass and overhang DNA synthesis.
Abasic sites are the most frequent DNA lesions formed in the genome149 and are the result of hydrolysis of the glycosidic bond that connects the nucleobase with the sugar moiety. It has been estimated that approximately 10000 abasic sites per day are formed in a human cell150. Most are sensed and excised by DNA repair systems using the sister strand to guide incorporation of the right nucleotide in the position of the lesion, but sometimes abasic sites stay undetected and cause problems in DNA replication. Abasic sites are strong barriers for replicative DNA polymerases. Native abasic sites are instable and may lead to strand breaks. That is why in most research studies the stabilized tetrahydrofurane analogue (Figure 38) is used. It has been shown in several studies that adenine is most frequently incorporated opposite of that lesion followed by guanine, a behaviour which is termed as ’A-rule’ 4,132. However, the mechanism that is responsible for purine selection by DNA polymerases is not fully understood.
Figure 38 Hydrolysis of the glycosidic bond leads to loss of the coding nucleobase and formation of an abasic site (left). Stabilised abasic site analogue (right).
Gloeckner et al.151 recently described an N-terminal shortened mutated Taq DNA polymerase (KlenTaq DM KK) from Thermus aquaticus which showed significantly
and M747K). In both cases hydrophobic amino acids (isoleucine and methionine, respectively) having van der Waals contacts with the backbone of the nucleotide or DNA substrate were substituted by a polar, positively charged lysine. KlenTaq DM KK was mainly characterized by radiometric primer extension analyses. It was found that KlenTaq DM is > 50 times more efficient in incorporating dATP nucleotides opposite the abasic site than the wild-type enzyme151.
Furthermore, it is known that 3’-5’-exonuclease deficient DNA polymerases usually add an additional nucleotide to a blunt-ended primer template complex in a non-templated manner121 preferentially incorporating dATP, thus producing a single stranded overhang at the 3´end (see Figure 39).
Figure 39 Blunt-ended primer template DNA complex. 3´-5´exonuclease deficient DNA polymerases add an additional nucleotide in a non-templated manner, preferentially dA121.
Intrinsic properties of adenine like superior stacking ability of the incoming adenosine nucleotide to the nucleobase system at the primer end as well as solvation were proposed as being the driving forces for preferential adenine selection. Until now, in reactions with blunt-ended or the abasic site substrate all known explanations for the preferential purine selection are still unconvincing.
Recently, Schnur et al.152 crystallised KlenTaq DM incorporating either an adenine or guanine opposite an abasic site substrate. Additionally, Obeid et al.153 was recently able to crystallise KlenTaq DM bound to a similar DNA duplex structure as depicted in Figure 39. In both situations it was found that a tyrosine (Y671) fills the space of the absent template nucleobase both at the abasic site and at the blunt-ended DNA substrate of the nascent nucleobase pair, thereby mimicking a pyrimidine nucleobase in shape and size.
2.5.2 Results
Inspired by the crystal structures obtained from Schnur and Obeid, in which a tyrosine Y671 fills the space of the absent template, KlenTaq wild-type and KlenTaq DM were
first tested in radiometric primer extension reactions for incorporation of the four different triphosphates (dATP, TTP, dCTP and dGTP, see Figure 40).
Figure 40 (A) Partial primer template sequence used in the dNTP incorporation opposite abasic site experiment. (B) Single nucleotide incorporation of KlenTaq wild-type and DM opposite abasic site at identical conditions at 72°C for 0.5, 2, or 10 min., respectively. The respective enzyme (50 nM) and dNTP (100 μM) are indicated.
Both enzymes were incubated separately with respective dNTPs at 72°C for increasing time periods (0.5, 2 and 10min). Indeed, the fastest incorporation opposite an abasic site analogue is found for dATP. Thus, wild-type and DM followed the A-rule and preferentially incorporated dA in a non-templated manner as expected. Furthermore, DM incorporated dATP much faster than the wild-type enzyme producing the 24 nt long product in only 30sec., which is in line with previous kinetic measurements151 that KlenTaq DM is > 50 times more efficient in incorporating dATP nucleotides opposite the abasic site than the wild-type enzyme.
Next, wild-type and DM was studied in reactions with a blunt-ended DNA substrate to study 3´ overhang synthesis (see Figure 39 and Figure 41). Again, both enzymes were incubated separately with respective dNTPs at 72°C for increasing time periods (0.5, 4, 15 and 60min; see Figure 41). As in the case of an abasic site substrate, both enzymes preferred incorporation of dA at the blunt-ended DNA substrate. KlenTaq wild-type produces mainly a 24nt long product in presence of dATP after 1h incubation time, whereas DM generates the same product after 30sec. of incubation. Interestingly,
Figure 41 (A) Partial primer template sequence used in the dNTP incorporation assay at ended DNA substrate. (B) Single nucleotide incorporation of KlenTaq wild-type and DM at the blunt-ended DNA substrate at identical conditions at 72°C for 0.5, 4, 15 and 60 min., respectively. The respective enzyme (100 nM) and dNTP (200 μM) are indicated.
Due to the finding that both at the abasic site and at the blunt-ended DNA substrate a tyrosine (Y671) fills the space of the absent template nucleobase of the nascent nucleobase pair (Schnur and Obeid152,153), amino acid sequence alignment was performed (see Figure 42). It was found that in all A-family DNA polymerases from prokaryotic and eukaryotic origins the corresponding O helices are highly conserved throughout evolution from bacteria to humans154. Especially the respective aromatic residue Y671 is evolutionary highly conserved.
Figure 42 Amino acid sequence alignment of DNA polymerases covering position Y671 from Thermus aquaticus Polymerase I.
To point out that Y671 plays the most important role for the A-rule behaviour in KlenTaq DNA Polymerase, two mutants of KlenTaq were constructed by site directed mutagenesis: The Y671A mutant was constructed in order to test the requirement of an aromatic residue at this position. The second mutant Y671W was constructed to explore a possible molecular mimicry mechanism in a way that the tyrosyl-side chain
Y671 of the DNA polymerase directs for incorporation of a purine opposite the missing template base (see Figure 43).
Figure 43 Chemical structures of purines (A,G) and pyrimidines (C,T) compared with tyrosine (Y) and tryptophane (W) demonstrating similar steric demands.
The mutation Y671W will transform the six-membered phenol ring in tyrosine into a bicyclic indole in tryptophane, consisting of a six-membered ring fused to a five-membered ring. If a mimicry mechanism is at work this would result in an approximate size of adenine or guanine and should thus mimic a purine residue instead of a pyrimidine nucleobase:
First, the resulting mutants were over-expressed in E. coli cells, purified by Ni-NTA affinity chromatography and visualised on SDS-PAGE to control for purity and concentration adjustment by comparing to a BSA standard dilution series (see Figure 44).
Figure 44 SDS-PAGE gel of purified KlenTaq DNA polymerases wild-type (wt), Y671A and Y671W.
Again, both mutants Y671A and Y671W were tested in radiometric primer extension reactions for incorporation of the four different triphosphates (dATP, TTP, dCTP and
Figure 45 (A) Partial primer template sequence used in the dNTP incorporation opposite abasic site experiment. (B) Single nucleotide incorporation of KlenTaq Y671A and Y671W opposite abasic site at identical conditions at 72°C for 2, 10 and 60 min., respectively. The respective enzyme (500 nM) and dNTP (100 μM)are indicated.
Indeed, mutation Y671A led to an enzyme that has significantly reduced activity in nucleotide incorporation both at the blunt-ended duplex and with the abasic site substrate (see Figure 45 and Figure 46, left side). Only very less product formation was observed after incubation with dGTP or dATP at 72°C for 60min and longer in both cases, whereas in the case of Y671W the preference for dA vanished and instead the pyrimidines TTP and dCTP are most efficiently incorporated (see Figure 45 and Figure 46, right side). Interestingly, during incubation of mutant Y671W with dCTP and TTP, a faint band indicating a 25 nt long product is also formed, which is similar to the findings with KlenTaq DM above.
Figure 46 (A) Partial primer template sequence used in the dNTP incorporation assay at ended DNA substrate. (B) Single nucleotide incorporation of KlenTaq Y671A and Y671W at the blunt-ended DNA substrate at identical conditions at 72°C for 4, 15,60 and 240 min., respectively. The respective enzyme (100 nM) and dNTP(200 μM) are indicated.
Kinetic studies with KlenTaq wild-type and Y671W with blunt-ended DNA duplex substrate (see Figure 46, (A), above) were performed to further evaluate the changed substrate preferences of the mutant Y671W from an A-rule to a T-rule behaviour (see Table 5).
Table 5 Nucleotide incorporation at the blunt-ended DNA duplex substrate
enzyme dNTP kpol [s-1·10-2]
Kd [μM]
kpol/Kd
[s-1·μM-1·10-4]
wt dATP 11.4 ± 0.86 240 ± 33.2 4.75
wt TTP 0.72 ± 0.15 838 ± 258 0.09
Y671W dATP 0.49 ± 0.06 630 ± 122 0.08
Y671W TTP 1.63 ± 0.31 934 ± 251 0.17
While dT is incorporated at the blunt-ended substrate by the wild-type enzyme with
~50-fold lower efficiency (kpol/KD) compared to dA, the opposite is observed for Y671W.
The mutant enzyme shows a 3-fold higher efficiency for dT incorporation compared with dA.
2.5.3 Conclusion
These findings corroborate the model of a nucleobase mimicry mechanism for the
the substrates to the active site of the enzyme for DNA polymerase activity. In accord with these observations, it has been shown that DNA polymerases are able to process non-natural nucleobase surrogates placed in template strands that mimic the shape and size of the natural nucleobase but have decreased hydrogen bonding capabilities155. Due to the nucleobase mimicry of Y671, purines more favourably fill the vacant space in the binding pocket than the smaller pyrimidines thymine and cytosine.
The preference for adenine over guanine incorporation opposite abasic sites (A-rule) can be rationalized by the superior stacking and solvation properties of adenine over guanine, as envisioned before155. Furthermore, the aromatic residue Y671 is evolutionary highly conserved. Amino acid sequence alignment of A-family DNA polymerases from prokaryotic and eukaryotic origins showed that the corresponding O helices and especially Y671 is highly conserved throughout evolution from bacteria to human. Due to the high conservation in DNA polymerases from prokaryotes, eukaryotes and archaea, it is likely that the depicted mechanisms of non-templated DNA synthesis are general and apply to other DNA polymerases in this sequence family as well. Especially in the case of abasic sites, the findings are of physiological relevance. The depicted mimicry of nucleobase shape and size has not been reported previously for a translesion synthesis. This mechanism to use protein residues to direct nucleotide incorporation is reminiscent to a transfer RNA CCA-adding enzyme156 and the specialized DNA polymerase Rev1 that exclusively incorporates cytosine nucleotides157. Other DNA polymerases such as of the Y-family are known to bypass several DNA lesions. However, they do not follow the A-rule in abasic site bypass4,132 and in contrast use the nucleotide 5’ to the abasic site as template. Thus, their selectivity cannot account for the A-rule observed in studies described above.
In summary, the purine specificity of non templated DNA synthesis, especially for the physiological very important abasic site translesion synthesis, in DNA polymerases from the A-family stems from specific interactions of the incoming dNTP with the protein side chain that mimics the size and shape of the absent nucleobase rather than the DNA.
A manuscript for publication of these results is currently under preparation.
3 Summary and outlook
In this PhD thesis, several projects about the functional analysis and recruitment of mutated DNA polymerases for improved biotechnological applications were investigated.
Discrimination of incorrect pairing single nucleotides is of fundamental importance for the enzyme-aided detection of single nucleotide variations (single nucleotide polymorphisms (SNPs)). It could be demonstrated that both chemically modified primer probes which are thiolated at the 2-position of thymidine as well as mutated DNA polymerases were able to increase single-nucleotide discrimination22.
Based on these findings a DNA chip based system for the multiplex detection of single nucleotide polymorphisms (SNPs) was established77. For that purpose, a mutated DNA polymerase from Pyrococcus furiosus with improved single nucleotide discrimination properties72 is used for selective microarrayed primer extensions. It is shown that the mutated DNA polymerase in combination with unmodified primer strands fulfils the demands on solid support and obviates the need for chemical modifications of the primer probes as required before22-24,74,76,104
. The system depicted herein could provide the basis for further advancements in microarrayed nucleic acid diagnostics using tailor-made enzymes.
Until now, all reported methods for DNA polymerase evolution are restricted to a single enzyme property, for example, increased selectivity or the ability to efficiently process DNA lesions28,31,39-44,46,50. Thus, a new microarrayed device was developed to overcome these obvious limitations that allow the multiplexed screening of several enzyme features in parallel:
The approach is based on the spatial separation of different covalently attached DNA substrates on a glass slide and their selective addressing by oligonucleotide hybridization. This system, termed oligonucleotide-addressing enzyme assay (OAEA), enables multiplexed simultaneous profiling of DNA polymerases in nanoliter volumes
other DNA-modifying enzymes like ligases and endonucleases can be included in multiplex directed evolution approaches using OAEA. As a first successful demonstration it was used to identify enzymes with altered properties out of a library of DNA polymerase mutants148.
A functional chimeric DNA polymerase could be obtained by fusion of a wild-type 5´-3´nuclease domain with a recently described N-terminally shortened DNA polymerase28 from Thermus Aquaticus, which exhibits a significantly increased reverse transcription activity. The new enzyme (named as Taq M1) was created to improve RNA pathogen detection systems for pathogens like Dobrava viruses. It could be demonstrated that the fusion of polymerase- and 3´nuclease-domain to constitute Taq M1 has no effect on the originally polymerase- and nuclease function and activities.
Additionally, Taq M1 was used in applied TaqMan RNA detection assays: Without optimisation of reaction conditions Taq M1 provided detection sensitivities compared to commercially available one-step RT PCR systems, which are based on enzyme blends.
Taq M1 is highly recommended for the use of one-step RT PCR, especially if high transcription temperatures are desired to melt stable secondary structures of RNA targets.
In my last project, functional studies were conducted with the N-terminally shortened DNA polymerase from Thermus Aquaticus (KlenTaq). Recently obtained crystal structures of KlenTaq in complex with both an abasic site harbouring template and a blunt-ended primer template substrate (Schnur et al.152 and personal communication with S.Obeid153), revealed that amino acid tyrosine 671 plays an important role in the template-less selection of the incorporated nucleotide. Tyrosine 671 thereby mimics the steric constraints of a pyrimidine template base resulting in the favoured incorporation of purine bases (A and G). Mutation of tyrosine into alanine (Y671A) results in a dramatic drop of catalytic activity. Mutation of the aromatic tyrosine into the also aromatic but steric more demanding tryptophane results in the favoured incorporation of pyrimidine bases (T and C). These findings could be proved by single nucleotide incorporation studies and enzyme kinetic measurements.
4 Zusammenfassung und Ausblick
In dieser Doktorarbeit wurden verschiedenste Projekte bearbeitet, die die funktionale Analyse und den Einsatz von mutierten DNA-Polymerasen in biotechnologischen, diagnostischen Anwendungen untersuchen:
Für die enzymatische Detektion von Einzelbasen-Varationen (SNPs, engl.: single nucleotide polymorphisms) ist die Diskriminierung von fehlgepaarten Einzelbasen von fundamentaler Bedeutung. Es konnte gezeigt werden, dass die Diskriminierung der fehlgepaarten Einzelbasen sowohl durch chemisch modifizierte Primersonden, die beispielsweise thiolierte Thymidine tragen22, als auch genetisch durch mutierte DNA-Polymerasen erhöht werden kann. Darauf aufbauend wurde unter anderem ein auf DNA-Chips basierendes Verfahren etabliert, welches die vielfache, synchrone Detektion von SNPs ermöglicht77. Hierbei konnte eine mutierte DNA-Polymerase aus dem Organismus Pyrococcus furiosus mit einer erhöhten Diskriminierungsfähigkeit gegenüber fehlgepaarten Einzelbasen72 erfolgreich eingesetzt werden. Das System aus mutierter DNA-Polymerase und unmodifizierten Primersträngen genügt der verlässlichen Detektion und ersetzt somit die Notwendigkeit des Einsatzes von chemisch modifizierten Primersträngen22-24,74,76,104
. Dieses Verfahren legt einen wegweisenden Grundstein für den Einsatz von maßgeschneiderten Enzymen in der modernen Nucleinsäure-Diagnostik.
Bisher sind alle bisher bekannten Methoden der gerichteten Evolution von DNA-Polymerasen auf eine einzige Enzymeigenschaft beschränkt, wie zum Beispiel der erhöhten Diskriminierungsfähigkeit gegenüber fehlgepaarten Einzelbasen
28,31,39-44,46,50
. Um diese deutliche Einschränkung zu umgehen, wurde ein Mikroarray-basiertes Verfahren entwickelt, das das vielfache Screenen von verschiedensten Enzymeigenschaften zeitgleich und parallel ermöglicht. Dieses Verfahren ermöglicht die selektive Hybridisierung von Oligonukleotiden durch kovalent befestigte und
können simultan verschiedenste DNA-Polymerase Eigenschaften in kleinsten Volumina (Nanoliter Mengen) im Hochdurchsatz gescreent werden. Zusätzlich besteht die Möglichkeit der Übertragung des Systems auf andere DNA-prozessierenden Enzyme wie Ligasen oder Nucleasen. Um die Praktikabilität zu demonstrieren, wurden in einer ersten Testreihe DNA-Polymerase-Mutanten mit gänzlich gegensätzlichen Eigenschaften, die aus einer kleinen Enzymbibliothek stammen, erfolgreich identifiziert und charakterisiert148.
Durch die Fusion einer Wildtyp 5´-3´Nuclease Domäne mit einer zuvor beschriebenen N-terminal verkürzten DNA Polymerase Mutante28 von Thermus Aquaticus, die eine signifikant erhöhte reverse Transkriptase Aktivität besitzt, konnte eine funktionale chimäre DNA Polymerase erhalten werden. Das neue Enzym (genannt Taq M1) wurde erzeugt, um eine verbesserte RNA-Pathogen Detektion, zum Beispiel von Dobrava- und Gelbfieber-Viren, zu ermöglichen. Es konnte gezeigt werden, dass die Fusion zur Taq M1 ohne Verlust von ursprünglicher Polymerase- oder Nuklease-Funktion bewerkstelligt wurde. Zudem wurde Taq M1 in angewandten TaqMan RNA Detektionsassays getestet: Ohne Optimierung an Reaktionsbedinungen lieferte Taq M1 vergleichbare Detektionsergebnisse wie kommerziell erhältliche one-step RT-PCR Systeme, welche auf den Einsatz von Enzymmischungen beruhen. Durch ihre Thermostablität empfiehlt sich Taq M1 somit für den Einsatz von one-step RT-PCR insbesondere dann, wenn erhöhte Transkriptionstemperaturen gewünscht sind, um besonders stabile Sekundärstrukturen des RNA Targets aufzuschmelzen.
In meinem letzten Projekt wurden funktionale Studien an der N-terminal verkürzten DNA-Polymerase von Thermus aquaticus (KlenTaq) durchgeführt. Durch zwei kürzlich erhaltene Kristallstrukturen der KlenTaq Polymerase sowohl mit einer abasischen Stelle- als auch einem blunt-end enthaltenen DNA-Templat konnte gezeigt werden, dass die Aminosäure Tyrosin671 eine wichtige Rolle in der templatlosen Auswahl des inkorporierten Triphosphates spielt (Schnur et al.152 und S. Obeid153 persönliche Kommunikation). Das Tyrosin imitiert den sterischen Anspruch einer auf der Templatstrangseite stehenden Pyrimidinbase, was im bevorzugten Einbau von Purinbasen (A und G) resultiert. Durch die Mutation des Tyrosins zu Alanin verliert das
Enzym stark an katalytischer Aktivität. Eine Mutation des Tyrosin zu dem sterisch anspruchsvolleren Tryptophan hingegen resultiert zu dem bevorzugten Einbau von nunmehr Pyrimidinbasen (T und C). Dies konnte durch funktionale Einzeleinbaustudien demonstriert und durch Kinetikmessungen untermauert werden.