Intrinsic properties of a KlenTaq DNA polymerase mutant with enhanced substrate specificity determined by X-ray structure analysis

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Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)

an der Universit¨at Konstanz

Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie

vorgelegt von

M.Sc., Dipl.-Ing. (FH) Andreas Schnur aus Spaichingen

Tag der m¨undlichen Pr¨ufung: 26.06.2009 1. Referent: Prof. Dr. Wolfram Welte

2. Referent: Prof. Dr. Andreas Marx

Konstanzer Online-Publikations-System (KOPS) URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-82735

URL: http://kops.ub.uni-konstanz.de/volltexte/2009/8273/

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1.2.1 DNA polymerases in vivo . . . 7

1.2.2 DNA polymerases in molecular biology . . . 8

1.3 Mechanism and structure of DNA polymerases . . . 9

1.3.1 The nucleotide incorporation pathway . . . 10

1.3.2 Two metal ion mechanism . . . 11

1.4 DNA polymerase I . . . 13

1.4.1 TaqDNA polymerase and theKlenTaq . . . 13

1.4.2 Motif C from theKlenTaq . . . 14

2 KlenTaqM747K - open form 17 2.1 Abstract . . . 17

2.2 Introduction . . . 18

2.3 Material and Methods . . . 20

2.3.1 Bacterial strains . . . 20

2.3.2 Expression and Purification . . . 20

2.3.3 Crystallization and data collection . . . 21

2.4 Results and discussion . . . 23

3 KlenTaqM747K with primer/template and ddCTP 29 3.1 Abstract . . . 29

3.3.1 Expression and Purification with His6-tag . . . 30 I

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3.4.1 KlenTaqM747K in ternary complex with p/t and ddCTP . . . 33

3.4.2 KlenTaqM747K in ternary complex with p/t, ddCTP and dCMeTP . . 40

4 KlenTaqM747K, I614K with primer/template and ddNTP 43 4.1 Abstract . . . 43

4.4.1 Details of the M747K mutation . . . 50

4.4.2 Details of the I614K mutation . . . 52

4.4.3 PDB Accession Code . . . 52

5 KlenTaqM747K, I614K with primer/abasic template and ddNTP 55 5.1 Abstract . . . 55

5.4.1 A protein tyrosine mimics the absent base of the abasic template . . . . 59

5.4.2 Details of the I614K mutation . . . 65

5.4.3 PDB Accession Code . . . 65

6 Summary 67 7 Zusammenfassung 71 8 General Materials and Methods 75 8.1 Materials and Chemicals . . . 75

8.1.1 Expression strains . . . 79

8.1.2 Enzymes and Proteins . . . 79

8.1.3 Nucleotides and Oligos . . . 79

8.1.4 Crystallization screens . . . 80 II

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8.4 Crystallographic methods . . . 86

8.4.1 Sitting drop . . . 86

8.4.2 Hanging drop . . . 87

8.4.3 Hyper quenching . . . 87

References 87 9 Appendix 105 9.1 Amino acids nomenclature . . . 105

9.2 Abbreviations . . . 106

9.3 Vector maps and sequences . . . 109

9.3.1 pASK-IBA37plus . . . 109

9.3.2 pGDR11 . . . 111

9.3.3 KlenTaqwild-type open reading frame . . . 113

9.4 The Nucleix Crystallization Screen . . . 115

9.5 Danksagung . . . 118

III

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Enzymes are an important class of proteins, which are essential for all metabolic processes in life. They are macromolecular machines which can act in different manners, for example as transporters, as ion pumps and as catalysts. They are able to catalyze reactions which cannot occur without them and can speed up chemical reactions with a factor of10⁸-10⁹. Enzymes, like all proteins, consist of large polymers made out of 22 different L-αamino acids, 20 stan- dard amino acids plus selenocysteine and pyrrolysine. The amino acids are joined together by peptide bonds between the carboxyl group of one amino acid and the amino group of the adjacent one. The linear sequence of successive amino acids is designated as primary structure, which contains the information of protein folding in the sense that it determines the minimal free energy conformation. The secondary structure describes the local folding of the backbone, such asα-helix,β-sheet and loops and the tertiary structure is the full 3-dimensional description of the molecule. Some proteins build oligomeric structures in a well-defined manner, such as homo- or hetero- dimers, trimers and higher order structures, which is understood as the quaternary form of the protein. The tertiary structure of a monomeric protein and the quaternary structure of an oligomeric protein are the active forms and the correct folding is responsible for the proper function of the protein. Highly selective reactions take place in the active center, which provides very well-defined conditions and is often conserved between homologous proteins with the same functions in different organisms. The surrounding condition, such as buffer, pH and temperature, influences the activity of the protein.^[1]

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1.1 DNA - The Deoxyribonucleic acid

1.1.1 Description of DNA

Apart from the enzymes, there is another essential macromolecule in cells - the Deoxyribonu- cleic acid, the DNA. DNA is the informational storage and the construction plan of all cells. It contains the genetically encoded information for proteins and RNA molecules. DNA is a poly- mer made up by concatenate small units called nucleotides. The molecular structure of the DNA was first discovered by James Watson and Francis Crick in 1953 with the help of X-ray diffraction images from Rosalind Elsie Franklin.^[2,3] The DNA is a double helix (Fig. 1.1) build from 4 different nucleotides connected to the adjacent nucleotide by a phosphor diester bond. The 4 possible nucleotides in the DNA are the purines (Adenine, A; Guanine, G) and the pyrim- idines (Thymine, T; Cytosine, C) bound to the 2-deoxyribose and the phosphate group. The accurate base pairing occurs between purine and pyrimidine bases (A:T; G:C) by Watson-Crick base-pairing (Fig. 1.2). Different conformations of the DNA, as illustrated in Figure 1.3, are

Figure 1.1: A schematic diagram of the Watson-Crick double helix. The two ribbons symbolize the two phosphate-sugar chains, and the horizontal rods the pairs of bases holding the chains together. The vertical line marks the fibre axis (adapted from Watson and Crick 1953^[2]).

possible and the most frequent conformation of the right-handed double-helical Watson-Crick model is the B-DNA (Fig. 1.3B).^[5] This canonical Watson-Crick structure is a right-handed double helix with ten nucleotides per turn, separated by a 3.4 ˚A translation along the helix axis.

The two chains are aligned in mutually antiparallel orientation (Fig. 1.3).^[5] The DNA strands wind around each other and leave gaps between each set of phosphate backbones, exposing the sides of the bases inside. Two grooves twist around the surface of the double helix: the major

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Figure 1.2: Watson-Crick base pairing. Adenine and thymine form two hydrogen bonds, whereas guanine and cytosine form three hydrogen bonds (adapted and modified from Huppert 2008^[4]).

groove, which is 22 ˚A wide and the 12 ˚A wide minor groove.^[6] The edges of the bases in the major groove are more accessible than those in the narrow minor groove. Therefore, double stranded (ds) DNA binding proteins, like transcription factors bind to specific sequences in the major groove, at the sides of the exposed bases.^[7] The A-DNA (Fig. 1.3A) was the first description of calf thymus DNA under low humidity. A-DNA is a right-handed double helix with 11 nucleotides per turn and the base pairs are displaced from the helix axis as well as being in- clined to it. The B-form which is closest to the original Watson-Crick model is observed under conditions of high relative humidity and is characterized by a near-perfect ’ten’ units per turn and the base pairs being located nearly astride the helix axis and normal to it.^[5] There exist intermediate states between conformations and another form is the Z-DNA (Fig. 1.3Z) a left- handed duplex structure with a dinucleotide repeat unit, generally confined to alternating purine (G) and pyrimidine (C) sequences. It has six dinucleotides per turn and exhibits a characteristic zigzag backbone. This is a consequence of distinctly different geometries for the two residues in the dinucleotide repeat which arise from alternating sugar puckers andsys/anticonformations about the glycosyl bond.^[8,9]

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Figure 1.3: The currently accepted fibre model structures for A-, B- and Z-DNA are shown here using the ball-and-stick representation (Chandrasekaranet al., 1989^[10], Chandrasekaran & Arnott, 1996^[11]; Arnottet al., 1980^[8]) The nucleotides are colour-coded (cytosine in yellow, guanine in cyan, thymine in green and adenine in red) and a ribbon is superposed on the backbones connecting the P atoms. A-DNA and B-DNA are both right-handed uniform double-helical structures, while Z-DNA is a left-handed double helix with a dinucleotide repeat and the backbone follows a zigzag path (adapted from Ghosh & Bansal, 2003^[5]).

1.1.2 DNA damage and repair

In human cells, DNA damage occurs every day. A rate of 10⁴-10⁶ damages per day and cell is estimated, depending on the environmental influences. Calculated for the whole adult human body which has10¹²cells,10¹⁶-10¹⁸DNA damages occur daily.^[12]Reasons for induction of DNA damages are the UV-radiation from sunlight, X-ray radiation, reactive oxygen species (ROS), chemically active substances and spontaneous hydrolysis.^[13,14] Figure 1.4 shows an overview of the most important agents which cause DNA damage, the lesion formed, and the repair pathways employed to remove these lesions from DNA.^[14] Hydrolysis is the simplest reaction that is potentially harmful to DNA.^[15] Abasic sites (Fig. 1.5[1]), which are estimated to arise almost 10 000 times per human cell per day,^[16]are very base-labile and can also fragment further spontaneously to form cytotoxic single-strand breaks. The abasic sites are products of depurination and have lost the genetic coding information and can, thus, lead to mutations

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Figure 1.4: Most common DNA-damaging agents, lesions, and repair pathways (adopted from Schaerer^{[ 14 ]}).

called ROS such as superoxide radical anions, hydrogen peroxide, or hydroxyl radicals are generated which can modify DNA. These by-products of oxygen metabolism can react with DNA and lead to one of over 100 oxidative modifications in DNA found to date.^[19–22] One example and the most prominent oxidative base adduct is the 8-oxoguanine (Fig. 1.5[3]), which is mutagen and can block transcription. Another example is thymine glycol (Fig. 1.5[4]), which is only mildly mutagen but blocks DNA replication and transcription.^[23,24] ROS can also modify the DNA bases in an indirect fashion, where for example, polyunsaturated fatty acids are readily oxidized to form bifunctional electrophiles such as malondialdehyde, which can lead to the formation of the pyrimidinopurinone adduct M1G (Fig. 1.5[5]) or epoxidation products of acrolein, which form exocyclic etheno modification of A (Fig. 1.5[6]), C and G.^[14,25,26] Co- factors of enzymatic reactions, such as S-adenosylmethionine (SAM), a methyl-group donor, can accidentally methylate DNA to form 7-methylguanine, a relatively harmless lesion, and 3- methyladenine (Fig. 1.5[7]), which is highly cytotoxic as a result of its ability to block DNA replication.^[14,27–29] O⁶-methylguanine (Fig. 1.5[8]) is only a minor product of methylation, but it is mutagenic and cytotoxic and therefore, a separate repair pathway has evolved to deal with this lesion.^[30]Main pathways for the repair are the base excision repair (BER) and direct repair which are involved in the repair of spontaneously damaged DNA caused by normal cel- lular metabolism.^[15,31,32]In addition to the endogenous sources, there are numerous exogenous sources of damage, e.g. UV radiation from sunlight. UV radiation leads to the formation of pho-

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Figure 1.5: Examples of common lesions to DNA. Hydrolysis induced: [1] Abasic site, [2] Uracil; Oxidative damage: [3] 8-oxoguanine, [4]

thymine glycol. Indirectly produced: [5] M1G, [6] 1,N⁶-ethenoadenine; endogenous and exogenous methylating agents form, among others, [7] 3-methyladenine, [8] O⁶-methylguanine. UV radiation induced: [9] cyclobutane pyrimidine dimers (CPD), [10] photoadducts. Aromatic amines or nitro compounds and aromatic hydrocarbons are metabolized in the cells to reactive intermediates that generate adducts of [11, 12]

upon reaction with DNA. Many antitumor agents form DNA adducts [13], cisplatin forms intra- and interstrand DNA crosslinks by linking two guanine bases in close proximity (adapted from Schaerer, 2003^[14]).

toadducts between adjacent pyrimidine residues in DNA, cyclobutane pyrimidine dimers (CPD) (Fig. 1.5[9]) and photoadducts (Fig. 1.5[10]).^[33] There are numerous additional examples of adducts of genotoxic compounds, e.g. those formed by environmental mutagens such as aromatic amines (Fig. 1.5[11]) and aromatic hydrocarbons (Fig. 1.5[12]) and by antitumor agents such as cisplatin (Fig. 1.5[13]). These compounds, also known as bulky adducts,^[14,34–36]lead to a strong local distortion of the DNA double helix and block transcription and DNA replication.

In mammals, these bulky adducts are removed by nucleotide excision repair (NER).^[31,37–39]The ability of NER to remove a large number of structurally diverse lesions from DNA may have evolved specifically to deal with lesions which are sporadically formed by environmental mutagens. Mammals remove pyrimidine dimers by NER, but lower eukaryotes, bacteria and plants are also able to remove these adducts by photolyases, which transform the dimers back into two

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responsible for replication, repair and recombination. In one of the best studied organisms Escherichia coli (E. coli), replicative DNA synthesis is catalyzed by the replisome, a multimeric protein complex^[41–43]shown in Figure 1.6. After initiation and unwinding of the dsDNA at oriC

Figure 1.6: Organization and dynamics of theE. colireplisome. Polymerase cycling at the replication fork. As the replisome advances, the clamp loader loads aβclamp on an RNA primer (pink) synthesized by DnaG (purple). When the lagging-strand polymerase replicates to a nick, it dissociates from DNA andβ(red) and cycles to a newly loadedβclamp. The figure was adapted from Johnson & O’Donnell, 2005^[42].

(replication origin), DnaB (a DNA helicase) unwinds the duplex DNA adenosine triphosphate (ATP) dependent in both directions. The resulting single-strands are stabilized by single-strand binding proteins (SSB-proteins). For the initiation of the DNA synthesis, a hybridised RNA- primer is necessary, which will be synthesized from the primosome (DnaB-DnaG complex), while DnaG is the DNA primase. With those primers, the replisome catalyzes DNA synthesis in 5’-3’ direction and the replication fork evolves. The replisome contains two multimeric DNA polymerase III subunits (Pol III), one for the leading and one for the lagging strand. Dur- ing synthesis, the SSB-proteins are displaced. At the leading strand, there is a continuous DNA synthesis in 5’-3’ direction, while the lagging strand can only be synthesized discontinuously in so called Okazaki-fragments.^[44]In complex with theβ-sliding clamp, Pol III increased the pro- cessivity 10-15 fold to more than 5000 nucleotides, compared to the unbound form (Fig. 1.6).

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The β-sliding clamp is a circular protein dimer that encompasses the dsDNA and increases the affinity of Pol III to the DNA. At the lagging strand, during discontinuous DNA synthesis, the complex must be removed after generating one Okazaki-fragment. This is done through the γ-clamp loader (Fig. 1.6), which opens the β-subunit and loads a new primer/template- complex.^[41]During the replicative DNA synthesis, the newly synthesized RNA-pieces remain as primers in the synthesized strand. An additional DNA polymerase, the E. coli DNA polymerase I removes the newly synthesized RNA-pieces with the 5’-3’ exonuclease activity. The mechanism starts with the binding of DNA polymerase I at the 3’-end of an Okazaki-fragment and the successive digestion of the following fragments, meanwhile the building fragment is elongated. This RNA to DNA translation is called the nick translation. The resulting DNA fragments are connected to a continuous DNA strand through DNA ligases.

1.2.2 DNA polymerases in molecular biology

DNA polymerases are widely used in numerous and central molecular biological methods and are key enzymes for amplification, analysis and modification of DNA. The most popular and important method which uses DNA polymerase was first described by Mullis^[45]- the polymerase chain reaction (PCR). Up to now, there are many specialized PCR based methods known, e.g.

PCR is applied for simple amplification of DNA sequences, production of modified dsDNA, quantification of pathogen DNA (Q-PCR),^[46,47] random mutagenesis of open reading frames (ORFs) for the generation of mutant libraries,^[48] forensic fingerprints,^[49] incorporation of flu- orescent dyes into cDNA libraries for generation of transcription profiling on microarrays^[50]

and detection of single nucleotide polymorphisms in different formats.^[51,52] Several applications benefit from mutated polymerases with altered functions that were isolated from different organisms^[53,54] and the optimization of thermostable polymerases by directed evolution. For example, mutants were designed with evolved higher thermostability^[55]or lower activity at low temperatures for an intrinsic hot start mechanism that leads to higher specificity in PCR amplifications.^[56]Other examples are polymerases with increased resistance to inhibitors, such as heparin, for directed analysis of blood samples^[55] and polymerases with increased error rates for more efficient and less biased random mutagenesis.^[57,58] Furthermore, thermostable polymerases were evolved by Marx and coworkers which display reverse transcriptase activity and are able to amplify damaged DNA without loss of fidelity, as well as another thermostable polymerase with enhanced substrate specificity.^[59,60] Another group from Holliger evolved an enzyme that allowed the generation of mixed RNA/DNA amplification products in PCR, demon- strating DNA and RNA polymerase as well as reverse transcriptase activity within the same enzyme.^[61] This evolved enzyme allows one-step amplification, without help from other enzymes, which could interact with each other. Furthermore, amplification of damaged DNA

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ciated with diseases. Moreover, a lot of the SNPs are connected to tolerance and susceptibility to drugs. This leads to personalized medicine, which can be custom-tailored for the genetic envi- ronment of the individuals.^[66]Analyses of SNPs include DNA polymerase based methods like selective primer extension reactions on microarrays as, e.g., minisequencing,^[67] the pyrosequencing technology^[68] or the allele-specific amplification (ASA).^[69,70] The minisequencing and pyrosequencing technologies rely on the insertion selectivity of DNA polymerases while ASA and selective mismatch- extension on microarrays^[71]depend on the ability to inefficiently extend mismatched primer/template complexes. Therefore, the reliability of ASA readouts can be significantly improved by enhanced selectivity of the used DNA polymerase in combination with modified nucleosides.

1.3 Mechanism and structure of DNA polymerases

As previously described, DNA polymerases are molecular motors which catalyze all occurring DNA synthesis from nucleotides. In organisms, the viability depends on the accurate replication of its genome. This is performed generally with only one error in every 10⁹-10¹⁰ bases replicated^[72]. Different mechanisms are responsible for this high accuracy. The initial discrimination occurs in the nucleotide incorporation stage (explained in the next section). During this initial discrimination, the DNA polymerase selects accurately a nucleoside triphosphate (dNTP) complementary to the template base provided by the template strand of DNA. Nevertheless, if the wrong nucleotide is connected by a phosphodiester bond to the primer and the polymerase contains an exonuclease activity, the 3’- 5’ exonuclease can remove this mispairing and the complementary nucleotide can be added subsequently. Additional error correction exists, namely the replicative excision and repair pathway.

Different models are available for the process by which DNA polymerases select the correct nucleotide. The first idea from Watson and Crick,^[2,73] based on the structure of DNA, was, that selection could be determined by the hydrogen bond-mediated base pairing between A-T and G-C. When comparing the error rates for nucleotide insertion of replicative polymerases (10⁻³-10⁻⁶ per replicated base) with the difference in energy between correct and incorrect

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base pairs, this would account for a higher error frequency (3·10⁻² per replicated base).^[74]

Therefore, the DNA polymerase must perform an active role, instead of acting like a zipper.

A later model proposed that the selection for correct or incorrect nucleotides depends on the base pair geometry.^[75–77]Hence, the shape of the active site is such, that a correct Watson-Crick base pair can fit perfectly, where a non-Watson-Crick base pair cannot fit in. Moreover, it has been proposed that free energy differences between correct versus incorrect base pairs could be amplified due to exclusion of water molecules in the polymerase active site.^[78]

Until now, there are several different polymerases known. For example, the prokaryote E. coli has five different DNA polymerases and eukaryotic cells have more than 12 different DNA polymerases.^[79] Based on the primary sequence homologies^[80–82] and crystal structure analyses,^[83]the different polymerases have been classified into seven different families: A, B, C, D, X, Y, and RT.^[84]

1.3.1 The nucleotide incorporation pathway

Different studies of DNA polymerases have established a minimal model of nucleotide incorporation for family A,^[85–91]family B,^[92,93]family X,^[94–97]family Y^[98,99]and family RT^[100–103]

polymerases.^[84]The minimal model for the nucleotide incorporation by all DNA polymerases is shown in Figure 1.7. Thereby, DNA polymerases catalyze the synthesis of DNA with an ordered mechanism in which the primer/template DNA (p/t) binds prior to the dNTP. The polymerization starts with the binding of a p/t to the unliganded enzyme (E) to form the enzyme-p/t complex (E:p/t) (Fig. 1.7[1]). Then the nucleotide (dNTP) incorporation into the E:p/t complex occurs to form the enzyme-p/t-dNTP complex (E:p/t:dNTP) (Fig. 1.7[2]). The following step, known as the rate-limiting step of polymerization, is the conversion of the E:p/t:dNTP complex to an activated complex, E’:p/t:dNTP (Fig. 1.7[3]). This activated complex is caused by a conformational change and can undergo the condensation reaction where the new 3’-5’ bond is formed. This chemical step starts with a nucleophilic attack of the 3’-OH primer terminus on theα-pyrophosphate of the dNTP, which results in the formation of a phosphodiester bond (Fig.

1.7[4]). This is followed by a second conformational change which results in the release of the pyrophosphate (PP_i) product (Fig. 1.7[5]). The DNA polymerase can either dissociate from the p/t (distributive synthesis) or translocate the p/t substrate for a new round of incorporation (processive). For the case that an incorrect nucleotide is incorporated, the new terminus can be translocated to the 3’-5’ exonuclease domain, if present, in order to be removed. Another pos- sibility is that the misincorporated nucleotide can be removed directly by pyrophosphorolysis (the reverse reaction of DNA synthesis), or the polymerase can extend the incorrect nucleotide,

“sealing” the misincorporated nucleotide within the elongated strand.^[84]

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Figure 1.7: Kinetic pathway of nucleotide incorporation. The various complexes are indicated with E = Enzyme, p = primer, t = template, PPi

= pyrophosphate. kpolis the rate constant of the rate-limiting step (adapted and modified from Rothwell & Waksman, 2005^[84]).

1.3.2 Two metal ion mechanism

The condensation reaction of 2’-deoxynucleoside triphosphate with 3’ end of the primer of the primer/template complex in the active center requires two divalent metal ions. For the accurate synthesis in the case of DNA polymerase I, two magnesium ions are necessary, which are coordinated by aspartates. In the error prone PCR, the magnesium ions are replaced with manganese ions, which leads to lower specificity and more mismatches. The proposed mechanism of nucleotide addition^[104] is in analogy to the reverse nearly identical mechanism of the 3’-5’ exonuclease of DNA polymerase I.^[105,106]One magnesium ion accompanies the incoming dNTP.

This magnesium ion is coordinated by the phosphates of the nucleotide and two conserved aspartates.^[107] The second magnesium ion is required to condensate the incoming nucleotide to the 3’-OH end of the primer by nucleophilic substitution under release of pyrophoshate. In detail, the first metal ion (Fig. 1.8[1]) lowers the affinity of the 3’-OH for the hydrogen, facilitating the 3’-O attack on theα-phosphate of the incoming dNTP. Both metal ions stabilize the structure and charge of the expected pentacovalent transition state and afterwards, the second metal ion (Fig. 1.8[2]) assists the leaving of the pyrophosphate.^[107,108]A third metal ion (Fig.

1.8[3]) which has not been observed until now, possibly may bind to theβandγ phosphate.^[104]

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Figure 1.8: Hypothetical chemical mechanism for the polymerase reaction based on two metal ion catalysis analogous to that proposed for the 3’-5’ exonuclease reaction from Beese and Steitz.^[105]It is proposed that the role of the catalytically essential carboxylates (Asp822, Glu883 and Asp705 in KF) is to bind two divalent metal ions as observed at the polymerase active site. One Mg²⁺[1] is hypothesized to promote the deprotonation of the 3’-OH of the primer strand analogous to the role of metal A^[105,106]in the exonuclease active site. The other Mg²⁺[2]

could stabilize the formation of the pentacovalent transition state at theα-phosphate by facilitating the formation of a 90^◦O-P-O bond angle. It could also facilitate the leaving of the pyrophosphate. A third Mg²⁺[3] may bind to theβandγphosphates (adapted from Steitz, 1993^[104]).

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by X-ray analysis.^[109–111]The DNA polymerase I consists of 928 amino acids with 3 functional subunits. The N-terminal region (amino acids 1-324) includes the 5’-3’ exonuclease domain, the amino acids 324-517 build the middle region and include the 3’-5’ exonuclease domain, and the C-terminal region (amino acid 521-928) includes the DNA polymerase function. Those three domains are relatively independent from each other with regard to their tertiary structure and their enzymatic activities.^[112,113] E. coliDNA polymerase I was digested by Klenowet. al with subtilisin in two fragments, the 35 kDa and 68 kDa fragment.^[114] The 68 kDa fragment, also known after his discoverer as the Klenow fragment (KF), has DNA polymerase function and 3’-5’ exonuclease activity. Some examples for the family A repair enzymes are theTher- mus aquaticusDNA polymerase I (Taq polymerase I), the Escherichia coli polymerase I (E.

colipolymerase I) and theBacillus stearothermophiluspolymerase I. Most of the polymerase I enzymes contain 5’-3’ exonuclease activity and 3’-5’ proofreading activity, while only the 5’-3’

exonuclease is required for viability, because it is necessary for the removal of RNA primers from Okazaki fragments generated during replicative DNA synthesis (see Chapter 1.2.1). The DNA polymerase activity is used to fill in the resulting gap. During repair, polymerase I enzymes also fill in DNA gaps that result from the removal of a variety of DNA lesions.^[40]

1.4.1 Taq DNA polymerase and the KlenTaq

The Taq DNA polymerase belongs to the repair enzymes of the family A polymerases.^[80] It is involved in nucleotide excision repair and in processing Okazaki fragments which are generated during lagging strand synthesis.^[115] The Taq DNA polymerase is descended from the thermophilic organismThermus aquaticusand has, therefore, the specific characteristic of thermostability. The half-life ofTaqDNA polymerase is 9 minutes at97.5^◦C. Due to this intrinsic property, theTaqDNA polymerase is used in the laboratory for the polymerase chain reaction (PCR).^[45] It is a monomeric enzyme with 832 amino acids and with a molecular weight of 94 kDa.^[116] Through subtilisin digestion, as mentioned in chapter 1.4, the 68 kDa Klenow fragment can be generated, known asKlenTaq. Several structural X-ray analyses ofKlenTaqshow, that the tertiary structure (Fig. 1.9), especially the polymerase domain, resembles the E. coli

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Figure 1.9: Ribbon diagram of the open binary protein-p/t (A) and closed ternary protein-p/t-ddNTP (B) complex of Klenow fragment from TaqDNA polymerase I (Klentaq1). The structure can be compared with an open right hand viewed on thepalm(magenta) with thefingers (green) and thethumb(blue) subdomain, as well as the bound primer (gray) and the template (turquoise). The N-terminal domain starts in the yellow subdomain, and the O helix is shown in red (2KTQ and 3KTQ, adapted from Liet. al., 1998^[124]).

DNA polymerase I.^[117–120] All polymerases have a common architectural framework consist- ing of three canonical subdomains termed thefingers,palmandthumbsubdomains as shown in Figure 1.9. The substrate for theTaqDNA polymerase is either a single stranded DNA (ssDNA) or a dsDNA. The catalytic activity and enzymatic capacity is determined by the pH-value, the KCl concentration as well as the concentration and kind of the essential divalent cations. The preferences for the essential divalent cations (in decreasing order) are: Mg²⁺, Mn²⁺, Co²⁺and Ni²⁺. TheTaqDNA polymerase exhibits a higher specific activity (units/mg) and a higher KM

value for dNTPs than the E. coli DNA polymerase I.[54,121–123] The mesophilic E. coli DNA polymerase I has the optimal reaction temperature between 22-37^◦Cand the thermophilicTaq DNA polymerase at 65-75^◦C.

1.4.2 Motif C from the KlenTaq

The accurateness of incorporation of the canonical nucleotides over noncanonical ones is important for biotechnological applications and depends on the intrinsic selectivity of the DNA polymerase. The crucial criteria for DNA polymerase selectivity is widely believed to be the size and shape of the active site.^[72]It is believed, that high-fidelity DNA polymerases edit the

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the nucleobase through the minor groove. Size augmentation by chemical modification of the 2’-deoxyribose of the 3’-terminal nucleotide results in a significantly decreased primer extension by family A DNA polymerases, which indicates that there are close contacts between the enzyme and the DNA substrate in this motif. Further, it was found that mutations in motif C can affect DNA polymerase mismatch- extension efficiency.^[127,128]Randomization of the three amino acids in motif C from the wildtype Gln782-Val783-His784 (QVH) led to active mutants with an increased mismatch-extension selectivity. This increased mismatch-extension selectivity was caused by a hydrophobic substitution from QVH to Lys782-Val783-Lys784 (LVL).^[128]

This is consistent with the replacement of the polar His to Ala, which results in the same ef- fect.^[127]

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Figure 1.10: A: Structure of the Klenow fragment fromTaqDNA polymerase I complexed with p/t and ddNTP in the closed conformation (PDB ID:3KTQ). Highlighted in black are six motifs that are conserved within family A DNA polymerases. B: Detailed view of motif C. The last nucleotide pair of the 3’-primer terminus and the incoming dNTP pairing to its cognate nucleobase in the template strand are shown (drawn by myself using 3KTQ and PyMOL^[129]and published in Strerath et al.^{[ 125 ]}).

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Selection of theKlenTaqby screening a library of arbitrarily randomized mutants for high activity in combination with lesion bypass activity leads to a mutant with a single cationic exchange in position 747 from the hydrophobic methionine in the wild-type to the cationic lysine in the M747K mutation.^[130]To gain further insights into the mechanistic origins, the structure of the mutant was determined by X-ray crystallography. Based on the crystallization protocol of the wild-type (PDB ID:1KTQ) from Korolevet al.^[120], crystallization conditions were tested. After initial difficulties, crystals of the open form ofKlenTaq M747K were obtained without DNA and could be analyzed by X-ray diffraction. Several diffracting crystals of the open form were tested on the ESRF in Grenoble, France and on the SLS in Villigen, Switzerland. Two independent crystallization conditions resulted in well diffracting crystals. The first condition was similar to the Tris HCl buffered condition of the open wild-type (PDB ID:1KTQ) with a pH between pH 9.3 and pH 9.6 and a second condition was similar to the HEPES buffered condition of the closed tertiary wild-type structure (PDB ID:3KTQ) with a pH between pH 7.3 and pH 7.8 (Fig. 2.4 and Fig. 2.6). The best protein crystal was obtained with the Tris HCl condition and diffracted to 3.0 ˚A in the monoclinic space group P2₁ with unit cell parameters of a= 94.0 A, b= 84.8 ˚˚ A, c= 258.8 ˚A,α=γ= 90.0 ˚ ,β= 90.1 ˚ . The structure was solved by Molecular Re- placement using the wild-type protein structure as target (PDB ID:1KTQ) and refined to R_{f ac}= 23.9; R_{f ree}= 27.6. Analysis of the refined structure shows that Nz from the lysine side chain of the single mutation (M747K) in the open form is relatively flexible. This is indicated by the low density around the Nz end of the lysine 747 side chain compared to the clear density of the main chain of this residue.

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2.2 Introduction

KlenTaq M747K was discovered by C. Gloecker^[130] by screening a library of arbitrarily randomized mutants. He found one out of 736 PCR active variants of KlenTaq which was able to bypass DNA lesions significantly more efficiently than the wild-type enzyme^[59](Fig. 2.1).

ThisKlenTaqcontains a single hydrophilic substitution from M to K at position 747. Moreover, KlenTaqM747K shows lesion bypass activity on stabilized abasic sites (Fig. 2.1A), 8-oxoA and 8-oxoG damaged templates (Fig. 2.1B) while the wild-type stops at those DNA damages. From

Figure 2.1: DNA synthesis with lesion bypass ofKlenTaqM747K. A) Experiments with the stabilized abasic site (AP). 5’-³²P-labeled primer- template complexes were incubated with equally high amounts ofKlenTaqwild-type (WT) andKlenTaqM747K (M747K), respectively and all four dNTPs. P: primer alone; A: template containing a dA residue at position marked with an X; AP: template containing a lesion at the position marked with an X. B) Experiments with oxidized purines. Reactions were conducted as described for (A) with the exception that oxidized lesions were introduced at position X (adapted from Gloeckneret al.^[59]).

structures of the wild-type (3KTQ) it is known that amino acid M747 is near the template DNA and is in contact with the 2‘-deoxyribose of the template of the previously incorporated base pair. Interestingly, the discovery of this mutation was not unique. There was another group, which found a substitution at this position in combination with a second substitution, namely E742K and M747K which is able to use RNA instead of DNA as template.^[131] Steady state measurements from C. Gloecker^[130] showed, thatKlenTaqM747K has a slightly lower selectivity for extension of mismatches compared with the wild-type. The relative efficiency for the extension of mismatch primers (dC/dT) and (dT/dT) is twice that for wild-type. Opposite a

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Figure 2.2: Generation of UV-damaged DNA and PCR amplification. A) Irradiation of double-stranded plasmid DNA with UV-light in varying doses. B) PCR amplification of an approximately 1.8-kb fragment using the UV-damaged DNA as the template. For all reactions identical conditions were employed in PCR. Left lane: DNA marker; right lane: PCR performed in the absence of target DNA. Analysis was performed by agarose gel electrophoresis and ethidium bromide staining (adapted from Gloeckneret al.^[59]).

where lesion bypass activity is reported for DNA polymerases, it is associated with relatively low specific activity and selectivity in processing non-damaged templates. However,KlenTaq M747K shows high activity and selectivity in combination with lesion bypass activity.^[130]Even the error rate for incorporation of the noncanonical nucleotide is of the same magnitude for the mutant compared with the wild-type (8.8x10⁻⁵ for the wild-type and 11.5x10⁻⁵ for the Klen- TaqM747K). In both cases it is typical for theTaqpolymerases that there are more transitions (purine-purine exchange [A^<-^>G] or pyrimidine-pyrimidine exchange [C^<-^>T]) than transver- sions (purine-pyrimidine, or pyrimidine-purine).^[132,133] Moreover, this mutant is able to use DNA that was damaged by UV-light as a target for PCR amplification contrary to the wild-type which is not able to proceed over such damages (Fig. 2.2A and B).^[130] UV-light damage pro- duces several different DNA damages (as mentioned in chapter 1.1.2). Most of these damages are pyrimidine dimers and bulky adducts. Those compounds normally block DNA replication

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and are removed by nucleotide excision repair.

Recently, DNA polymerases were discovered which are able to make trans-lesion synthesis (TLS). Those TLS polymerases are grouped in the Y-family polymerases.^[134–139] This makes them useful for PCR amplifications for damaged DNA and forensic applications. To understand how this single mutation interacts in theKlenTaqM747K and leads to the discussed differences in substrate tolerance and influence on the dNTP incorporation, the atomic structure of the mutant was determined by X-ray crystallography.

2.3 Material and Methods

2.3.1 Bacterial strains

TheE. coliBL21-Gold(DE3) was used for heterologous overproduction of theKlenTaqM747K.

This expression system, harboring the KlenTaq M747K sequence in the pASK37plus vector, purchased from IBA was taken over from C. Gloeckner.^[130] Between the N-terminal His₆- tag and the protein-coding sequence is a Factor Xa cleavage site in the pASK37plus. The vector contains a tet promoter inducible with anhydrotetracycline for cytoplasmic expression.

A second expression system was used inE. coliBL21-Gold(DE3) with the plasmid pGDR11 (a pQE31 (Qiagen) derivative harboring thelac^Iq gene^[140] obtained from J. Nesper) with a N- terminal His6-tag. Expression of the pGDR11 is under T5- promoter control of the phage T5 and regulated through the lac promoter.

2.3.2 Expression and Purification

Heterologous expression inE. coliBL21-Gold(DE3) was done in 10 x 0.8 l scale Luria Bertani (LB) medium (10 g/l tryptone, 5 g/l yeast extract, 10 g/l NaCl, pH 7.0) with 100 µg/ml car- benicillin for pASK37plus or with 100 µg/ml ampicillin for the pGDR11. The medium was inoculated with 1:100 (v/v) overnight culture and cells were grown at 37^◦Cin a shaking in- cubator at 220 rpm. After cell growth reached the mid-exponential phase (OD600= 0.5-0.8), protein expression was induced with anhydrotetracyclin (AHT) in the pASK37plus to an end- concentration of 200µM and with 1 mM IPTG in the pGDR11. After 4 h expression, the cells were harvested by centrifugation at 5000 x g for 10 min. Subsequently, pelleted cells were lysated for 30 min at 35^◦C and 250 rpm using 30 ml lysisbuffer (10 mM Tris HCl pH 9.0, 300 mM NaCl, 2.5 mM MgCl₂, 0.1 % Triton X100, 1 mM Benzamidin, 1 mM PMSF, 2 mg/ml lysozyme) per l cell culture. The lysate was heat denatured at 75^◦Cfor 50min to precipitate heat labile enzymes. After cooling down to room temperature, the solution was ultracentrifuged at 50 000 x g for 1 h. The supernatant was filtered through a sterile filter (cut off size: 50µm)

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Figure 2.3: SDS-PAGE of expression and Ni-purification ofKlenTaqM747K. Arrow indicates position of the purified protein. M: Marker M12;

1: before induction; 2: 1 h expression; 3: 3 h expression; 4: before induction; 5: 1 h expression; 6: 3 h expression; 7: after heat denaturation;

8: Supernatant of Ni-slurry 9: first Ni-slurry washing step; 10: second Ni-slurry washing step; 11: elution with 0.5 M imidazole.

from the Ni affinity purification was concentrated to 2 ml and applied to a pre-equilibrated (50 mM Tris HCl, 20 mM MgCl2, pH 9.0) gel filtration Superdex 200 (160 ml, GE Healthcare) to remove the imidazole and change the buffer. Pure protein elutes after 70 ml and the protein was concentrated to 7 mg/ml using centrifuge filtration devices (Vivaspin 50 kDa, Vivascience).

Expression could be optimized using vector pGDR11 (Chapter 9.3.2) instead of using the lower yield vector pASK37plus. In detail, out of 8 l cell culture, approximately 12 mg protein could be achieved with pGDR11 with the same purity as the 3 mg yield from pASK37plus.

2.3.3 Crystallization and data collection

Crystallization trials were carried out. Therefore, the protein was concentrated to 7 mg/ml and 20 mg/ml and crystallization screens were prepared using rational design based on the condition of the open 1KTQ^[120] form. A screen was prepared using the published condition (100 mM Tris HCl, pH 9.0, 50 mM MgCl₂, 6 % (w/v) PEG 3350) by varying pH from pH 8.2 to pH 9.5 (Fig. 2.4). First crystals were achieved with a protein concentration of 20 mg/ml in the hanging drop method using 100 mM Tris HCl, pH 9.5, 50 mM MgCl2, and 12 % PEG 3350 as reservoir (Fig. 2.4A). In fact, this condition is at the end of the screen so a second refined rational design

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screen (Fig. 2.4B) was prepared with extended steps regarding PEG 3350 concentration and with different MgCl₂ concentrations. In all conditions of the second screen appeared crystals after one week with a suitable size of 300 x 80 x 10 µm (Fig. 2.5B, C) in a hexagonal plate shape. Additionally, another condition from Li et al. 2001^[141] for the tertiary form (0.1 M Hepes, pH 7.5, 20 mM MnCl₂, 0.1 M Na-Acetate, 10 % (w/v) PEG 4000) was tested and a screen was prepared (Fig. 2.6A).

1 2 3 4 5 6

pH 8.2 pH 8.5 pH 8.7 pH 9.0 pH 9.3 pH 9.5

100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris A 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2

4% PEG3350 4% PEG3350 4% PEG3350 4% PEG3350 4% PEG3350 4% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris B 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2

6% PEG3350 6% PEG3350 6% PEG3350 6% PEG3350 6% PEG3350 6% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris C 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2

9% PEG3350 9% PEG3350 9% PEG3350 9% PEG3350 9% PEG3350 9% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris D 50mM MgCl₂ 50mM MgCl₂ 50mM MgCl₂ 50mM MgCl₂ 50mM MgCl₂ 50mM MgCl₂

12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350

A

1 2 3 4 5 6

pH 9.4 pH 9.4 pH 9.5 pH 9.5 pH 9.6 pH 9.6

100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris A 20mM MgCl₂ 50mM MgCl₂ 20mM MgCl₂ 50mM MgCl₂ 20mM MgCl₂ 50mM MgCl₂

12% PEG3350 10% PEG3350 12% PEG3350 10% PEG3350 12% PEG3350 10% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris B 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2 50mM MgCl2

12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 12% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris C 75mM MgCl2 50mM MgCl2 75mM MgCl2 50mM MgCl2 75mM MgCl2 50mM MgCl2

12% PEG3350 14% PEG3350 12% PEG3350 14% PEG3350 12% PEG3350 14% PEG3350 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris 100mM Tris D 100mM MgCl2 50mM MgCl2 100mM MgCl2 50mM MgCl2 100mM MgCl2 50mM MgCl2

B

Figure 2.4: Example of a rational design crystallization screen: [A] Initial and [B] refined crystal screen fromKlenTaqM747K. Crystals appeared in A in condition D6 and in all conditions in B.

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Figure 2.5: Crystals fromKlenTaqM747K (crystal plate 072), A: first hit at 100 mM Tris HCl pH 9.5, 50 mM MgCl2, 12 % PEG 3350, Size:

160µm x 70µm, B: Refinement screen of condition A: Size: 800µm x 300µm, Condition: 100 mM Tris HCl, 20 mM MgCl2, 12 % PEG 3350, pH 9.6. Space group P21, 3.0 ˚A, a=93.1 ˚A, b=84.1 ˚A, c=256.1 ˚A;α=γ= 90.0˚ ,β = 98.8˚ , 6 monomers per ASU C: Different crystals of the refinement screen, cracked on the down left hand side.

To prevent the crystals from secondary radiation damage, cryocrystallography is used and therefore, the crystals were frozen in liquid nitrogen and measured at 100 K. To avoid water crystallization, which would lead to ice rings and damage the crystal lattice, different cryopro- tectans were tested:

• Glycerol

• Ethyleneglycol

• PEG 300

• PEG 400

• PEG 550

• Propanediol

• Glycerol/Sucrose Mixture (60/40; 50/50; 40/60)

2.4 Results and discussion

Cryoconditions had to be established, where water at 100 K is in vitreous state so that it does not diffract the X-rays, while the protein crystal is still well ordered. This was tested by using a ro- tating anode generator (Schneider, Offenburg, Germany) equipped with a Proteum Pt¹³⁵detector (Bruker AXS, AG Welte). Sequential soaking of the crystals with reservoir solutions containing increasing glycerol concentration was tried. In the optimized concentration, the concentration was increased in 5% (w/v) steps until 20 % (w/v) glycerol were reached. Different crystals of theKlenTaqM747K were measured either on beamline ID14-4, ID23-2 or ID29-1 on the ESRF in Grenoble, France or the beamline X06SA at the SLS in Villigen, Switzerland. In Figure 2.7, the diffraction pattern of one frame from theKlenTaq M747K is shown, which diffracted to 2.7 ˚A on the beamline ID29-1 ESRF, in Grenoble. Data reduction was done with XDS^[142],

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1 2 3 4 5 6

pH 7.3 pH 7.4 pH 7.5 pH 7.6 pH 7.7 pH 7.8

0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc A 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2

8% PEG4000 8% PEG4000 8% PEG4000 8% PEG4000 8% PEG4000 8% PEG4000 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc B 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2

10% PEG4000 10% PEG4000 10% PEG4000 10% PEG4000 10% PEG4000 10% PEG4000 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc C 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2

12% PEG4000 12% PEG4000 12% PEG4000 12% PEG4000 12% PEG4000 12% PEG4000 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M Hepes 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc 0.1M NaAc D 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2 20mM MnCl2

A

B

Figure 2.6: A: Crystal Screen fromKlenTaqM747K, Hepes condition (0.1 M Hepes, 0.1 M Na-Acetate (NaAc), 20 mM MnCl2, 10 % (w/v) PEG 4000, pH 7.3) , B: Crystal ofKlenTaqM747K in Hepes condition from A, condition B1 (0.1M Hepes, 0.1M Na-Acetate, 20mM MnCl2, 10 % (w/v) PEG 4000 pH 7.3), Space group P21, 2.8 ˚A, a= 93.2 ˚A, b= 84.7 ˚A, c= 130.7 ˚A,α=γ= 90.0˚ ,β= 101.5˚ , 3 monomers per ASU.

data analysis using the CCP4i interface.^[143]The structure was solved with Molecular Replace- ment using Molrep^[144–147] and Phaser^[148] using 1KTQ^[120] as target for phase determination, Refmac^[149–153] for refining and Coot^[154] for model building and PyMOL^[129] for illustrations.

The space group was P21 with the unit cell: a=94.0 ˚A, b=84.8 ˚A, c=258.8 ˚A, α= 90.0 ˚ , β=

90.1 ˚ , γ= 90.0 ˚ . The refinement led to R_{f ac}= 23.9 % and Rf ree= 27.6 % with 6 molecules (A-F) per asymmetric unit (ASU). A non-crystallographic two-fold symmetry (NCS) between 3 monomers could be detected between A=D (293-731; 732-775; 776-832), B=E (293-431;

432-477; 478-832) and C=F (293-731; 732-775; 776-832) and was used for refinement. Due to the fact, thatβ is close to 90 degrees, other space groups were possible (16 (P222), 17 (P222₁), 18 (P2₁2₁2) and 19 (P2₁2₁2₁)) but the refinement could not decrease R_{f ree}below 34 % in these alternative space groups only in P21with NCS, the Rf reecould be decreased to 27.6 %. The best crystal diffracted to 2.7 ˚A at beamline ID23-2 on the ESRF, but due to twinning and radiation

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Figure 2.7: Diffraction pattern at the ID29-1, ESRF, Grenoble ofKlenTaqM747K to 2.7 ˚A. In the inset the frozen crystal mounted in a loop is shown.

damage, this dataset could not be processed. However, it was possible to solve the structure with another 3.0 ˚A dataset. Moreover, a second condition was found after screening the condition for the closed ternary structure (Hepes condition). This condition led to crystals with a resolution of 2.8 ˚A, and a size of 200 x 300µm (Fig. 2.6B). Data collection statistics are given in Table 2.1. Table 2.1 shows a dataset from the Tris HCl condition and a dataset of the Hepes condition. Due to high mosaicity, the Hepes condition could only be refined to R_cryst= 26.8 % and a R_{f ree}= 36.5 %. The structure ofTaqDNA polymerase M747K is shown in Figure 2.8. In this figure, the protein is shown in cartoon, while the K747 residue is shown in sticks. Due to unresolved density, the loop between amino acid 504 and amino acid 512 (Fig. 2.8 on the top right hand side near motif 1 - motifs are shown in Fig. 1.10) could not be built in the model, so this region seems to be very flexible in the structure. Comparison of the overall structure of the open form ofKlenTaqM747K (green) with 1KTQ (gray)^[120]is shown in Figure 2.9A. The core root mean square deviation (rmsd) between 1KTQ and theKlenTaqM747K open form is 1.162 ˚A. Significant differences in single motifs or loops could not be detected and the overall structures are very similar. In Figure 2.9B, a detailed view around methionine 747 from 1KTQ (in gray) and the mutated lysine 747 fromKlenTaq M747K is shown. The orientation of the side chain differs, but there was no clear density for the end of the side chain (NZ) compared with the clearly resolved density for the main chain. That indicates, that in the open structure, the residue K747 is not rigid and in the case where no DNA is bound to the protein, different conformations of this side-chain are possible. Simultaneously, during refinement of the crystals of the open form, crystallization trials with DNA and protein were made, which is described in

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the next chapter (chapter 3). Following the results that the side chain is not well ordered in the open structure, the main focus was now to get the structure of the DNA protein complex, to see interactions of this side chain and hopefully a well defined density in the case that lysine 747 interacts with the DNA.

Data collection

KlenTaqM747K Tris HCl condition

KlenTaqM747K HEPES condition

Beamline SLS X06SA SLS X06SA

Wavelength [ ˚A] 0.98024 0.9801

Resolution range [ ˚A] 50.0-3.0 50-2.8

Outer shell [ ˚A] 3.00-3.18 2.80-2.97

Rmeas[%]^∗ 12.5 (48.6) 9.5 (63.6)

Unique reflections 76058 49270

Total reflections 288312 184277

Mean(I/σ)^∗ 9.6 (2.4) 13.2 (2.5)

Completeness [%]^∗ 97.2 (83.8) 99.0 (95.9)

Space group P21 P21

Cell dimensions a,b,c [ ˚A] 94.0, 84.8, 258.8 93.3, 84.8, 130.7 Cell dimensionsα, β, γ[ ˚ ] 90.0, 90.1, 90.0 90.0, 101.5, 90.0

R_cryst[%] 23.9 26.8

R_{f ree} [%] 27.6 36.5

r.m.s. deviation bond length [ ˚A] 0.014 0.016 r.m.s. deviation bond angle [ ˚A] 1.865 1.894

Average B factors [ ˚A²] 51.7 48

Max. likelihood estimate for co- ordinate error [ ˚A]

0.420 0.470

Table 2.1: Data collection statistics for openKlenTaqM747K. Two representative datasets are shown here, a dataset from the Tris HCl condition and a dataset from the Hepes condition. The unit cell is in the Tris HCl condition twice the cell of the Hepes condition. In the Tris HCl condition are 6 monomers per ASU, whereas in the Hepes condition are 3 monomers per ASU, respectively. The smaller cell could not be refined further than Rcryst= 26.8 % and Rf ree= 36.5 %. Rmeas= redundancy independent R-factor (intensities) (for definition of Rmeassee Diederichs &

Karplus (1997), Nature Struct. Biol. 4, 269-275)^∗Values in parentheses correspond to those in the outer resolution shell.

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Figure 2.8: Structure ofKlenTaqM747K at 3.0 ˚A, Rf ac= 23.9 %, Rf ree= 27.6 %. Arrow indicates the lysine 747 mutation which is shown as sticks, while the other amino acids are shown in cartoon. The inset in the center shows the density (2Fo-Fc) of the lysine 747 side chain as a blue mesh at 1.5σ. This figure was created with PyMOL^[129]and the electron density map with Coot^[154].

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Figure 2.9: A: Overlay of the structure ofKlenTaqM747K (green) with 1KTQ (gray). Arrow shows the mutated lysine residue inKlenTaq M747K (green) and the methionine residue in theKlenTaqwild-type (1KTQ) in stick representation. The rest of the structure is shown in cartoon. B: Detail around lysine 747 (green) ofKlenTaqM747K and methionine 747 (gray) of theKlenTaqwild-type (1KTQ). Figure was created with PyMOL^[129].

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3.1 Abstract

The crystallization of theKlenTaqM747K in the open form was successful (previous Chapter 2). The results showed that the mutated lysine 747 side chain is flexible in the unbound form and has different conformations. To trap one conformation and resolve the interaction with DNA, crystallization trials of a tertiary complex with bound primer/template (p/t) and trapped with ddCTP were prepared. However, neither the use of the high expression vector pGDR11 nor the use of the low expression vector pASK37plus for protein expression inE.coliBL21-Gold(DE3) yielded protein that crystallized in tertiary form. The only attempt which led to crystals in the tertiary complex form, was by seeding crystals of the open form in a supersaturated crystallization condition with protein, DNA and ddCTP. Those crystals diffracted poorly to 5 ˚A. But these crystals could be reproduced and refined by seeding to a maximum resolution of 4.5 ˚A. To resolve interactions between DNA and mutation 747, this resolution was too low. As a new attempt, the His₆-tag was cleaved. Due to the fact that pASK37plus includes a Factor Xa cleavage site, protein expressed in the low expression vector pASK37plus in BL21-Gold(DE3) was used for crystallization without His₆-tag. After cleaving the His₆-tag with Factor Xa protease, high quality crystals of the tertiary complex were obtained with bound p/t and trapped with ddCTP.

The structure could be solved to a resolution of 1.8 ˚A with Rwork= 18.7 % and Rf ree= 24.1 % in space group P3121.

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3.2 Introduction

Crystallization trials of KlenTaq M747K with a 16mer template and an 11mer primer DNA (chapter 3.3.3) trapped with ddCTP (Fig. 3.1) were conducted similar to Liet al.^[141]. In the unbound open form it was possible to get well diffracting crystals (previous chapter 2), while in the closed form, bound to DNA, even with the same batch of purified protein, which crystallized in the open form, no crystals appeared. Several tests, regarding concentration, purity, homogeneity (dynamic light scattering) and activity were done, but no explanation was found why the tertiary complex did not crystallize. In one attempt, a crystal of the unliganded open form was seeded into the crystallization of the closed ternary complex. Interestingly, a conformational transition occurs and crystals of the closed form could be obtained (Fig. 3.3). However, these crystals diffracted to 5 ˚A only and reproduction was only possible by seeding. By seeding with a crystal of the tertiary complex into the new crystallization trial, the resolution could be increased to 4.5 A. Several additional tests did not lead to an improvement of the diffraction. A possible ob-˚ struct for crystallization could be the His6-tag, which can disturb crystal contacts in the protein or disturb the crystallization with DNA. Crystallization publications^[155,156] which compared His-tagged proteins and proteins without a His-tag showed that in most of the cases the His-tag has a negative influence on the crystallization behavior. This was the reason for changing the expression system back to the pASK-IBA37plus vector which has a Factor Xa cleavage site for cleaving the His₆-tag. Crystallization trials with p/t, ddCTP andKlenTaqM747K with digested His₆-tag were carried out.

Figure 3.1: Dideoxycytosintriphosphate (ddCTP) is used for trapping the DNA in theKlenTaq. The lack of the 3’-OH group compared with the natural DNA substrate deoxycytosintriphosphate (dCTP), leads after incorporation of one ddCTP to a trapped state, where no nucleophilic attack from the 5’-end of the triphosphate to the 3’- end of the primer is possible.

3.3 Material and Methods

3.3.1 Expression and Purification with His

₆

-tag

Expression and purification of the His6-tag containingKlenTaqM747K was similar as described under 2.3.2.