Characterization and application of a family B DNA polymerase from the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans Likui Zhang, Donghao Jiang, Haoqiang Shi, Mai Wu, Qi Gan, Zhihui Yang, Philippe Oger To cite this version: Likui Zhang, Donghao Jiang, Haoqiang Shi, Mai Wu, Qi Gan, et al.. Characterization and appli- cation of a family B DNA polymerase from the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans. International Journal of Biological Macromolecules, Elsevier, 2020, 156, pp.217-224. 10.1016/j.ijbiomac.2020.03.204. hal-02526997 HAL Id: hal-02526997 https://hal.archives-ouvertes.fr/hal-02526997 Submitted on 17 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 1 2 3 4 Characterization and application of a Family B DNA polymerase from the 5 6 7 hyperthermophilic and radioresistant euryarchaeon Thermococcus 8 9 gammatolerans 10 11 Likui Zhang1,4#, Donghao Jiang1, Haoqiang Shi1, Mai Wu1, Qi Gan1, Zhihui Yang2#, 12 13 3# 14 and Philippe Oger 15 16 17 18 1 19 Marine Science & Technology Institute, College of Environmental Science and 20 21 Engineering, Yangzhou University, Jiangsu Province 225127, China 22 23 2College of Plant Protection, Agricultural University of Hebei, Baoding City, Hebei 24 25 26 Province 071001, China 27 28 3Univ Lyon, INSA de Lyon, CNRS UMR 5240, Villeurbanne, France 29 30 4 31 Guangling College, Yangzhou University 32 33 #Corresponding author: Dr. Likui Zhang 34 35 Tel: +86-514-89795882 36 37 Fax: +86-514-87357891 38 39 E-mail address: [email protected] 40 41 Corresponding author: Prof. Zhihui Yang 42 43 44 E-mail address: [email protected] 45 46 Corresponding author: Prof. Philippe Oger 47 48 49 E-mail address: [email protected] 50 51 52 53 54 55 56 57 1 58 59 60 61 62 63 Abstract 64 65 66 Thermococcus gammatolerans is anaerobic euryarchaeon which grows optimally 67 68 at 88oC and its genome encodes a Family B DNA polymerase (Tga PolB). Herein, we 69 70 cloned the gene of Tga PolB, expressed and purified the gene product, and 71 72 73 characterized the enzyme biochemically. The recombinant Tga PolB can efficiently 74 75 synthesize DNA at high temperature, and retain 93% activity after heated at 95oC for 76 77 78 1.0 hr, suggesting that the enzyme is thermostable. Furthermore, the optimal pH for 79 80 the enzyme activity was measured to be 7.0-9.0. Tga PolB activity is dependent on a 81 82 divalent cation, among which magnesium ion is optimal. NaCl at low concentration 83 84 85 stimulates the enzyme activity but at high concentration inhibits enzyme activity. 86 87 Interestingly, Tga PolB is able to efficiently bypass uracil in DNA, which is distinct 88 89 90 from other archaeal Family B DNA pols. By contrast, Tga PolB is halted by an AP 91 92 site in DNA, as observed in other archaeal Family B DNA polymerases. Furthermore, 93 94 Tga PolB extends the mismatched ends with reduced efficiencies. The enzyme 95 96 97 possesses 3′-5′exonuclease activity and this activity is inhibited by dNTPs. The DNA 98 99 binding assays showed that Tga PolB can efficiently bind to ssDNA and primed DNA, 100 101 102 and have a marked preference for primed DNA. Last, Tga PolB can be used in routine 103 104 PCR. 105 106 Keywords: Archaea; DNA polymerase; DNA replication; Routine PCR 107 108 109 110 111 112 113 114 115 116 2 117 118 119 120 121 122 Introduction 123 DNA polymerase (pol) is a ubiquitous enzyme that replicates DNA in all living 124 125 126 cells. Since the first DNA pol was discovered and characterized from Escherichia coli 127 128 in the 1950s by Arthur Kornberg, a large number of DNA pols have been 129 130 131 biochemically and structurally characterized from diverse organisms. Currently, DNA 132 133 pols have been grouped into six families: A, B, C, D, X, and Y, based on amino acid 134 135 sequence similarities [1]. In addition, reverse transcriptase has been proposed as the 136 137 138 seventh family since it can synthesize DNA [2]. Family B DNA pols have been found 139 140 in bacteria, archaea, eukarya, and viruses/phages. A typical characteristic of most of 141 142 143 Family B DNA pols is that they possess 3′-5′ exonuclease activity in addition to 144 145 polymerase activity [3; 4], which can remove a misincorporated nucleotide opposite a 146 147 template strand, thus enhancing fidelity approximately 100-fold. 148 149 150 As the third life, Archaea are a simplified version of eukarya with respect to 151 152 DNA metabolism [5; 6; 7]. Currently, DNA pols of Family B, D, and Y have been 153 154 155 found in Archaea [8]. Family B DNA pols are ubiquitous in both Euryarchaeota and 156 157 Crenarchaeota, which are two major branches of Archaea. In addition to possessing 158 159 five conserved motifs, archaeal Family B DNA pols have a unique uracil binding 160 161 162 motif [9; 10], which can recognize uracil and hypoxanthine bases derived from 163 164 deamination in template DNA. Replication of uracil and hypoxanthine before repair 165 166 167 leads to GC to AT mutation and AT to GC mutation, respectively. However, 168 169 replication by archaeal Family B DNA pols would be stalled if deaminated base is 170 171 encountered with these pols [11; 12]. Such an error avoidance mechanism would be 172 173 174 expected to be evolutionary favorable for hyperthermophilic Archaea thriving in high- 175 3 176 177 178 179 180 181 temperature environments where the rates of deamination are increased. Hence, the 182 183 184 recognition of these pro-mutagenic lesions and the following replication stop 185 186 observed in archaeal Family B DNA pols might be a specific adaptation of 187 188 hyperthermophilic Archaea to their hostile environments. 189 190 191 As an important branch of euryarchaea, Thermococcus thrives in high- 192 193 temperature environments, including deep-sea hydrothermal vents and hot springs. 194 195 196 Increasing numbers of Thermococcus species have been isolated and identified [13]. 197 198 Since the first archaeal Family B DNA pol was reported from Thermococcus litoralis 199 200 [14], a dozen of archaeal Family B DNA pols have been biochemically characterized 201 202 203 from Thermococcus species: Thermococcus 9°N-7 [15], Thermococcuscelericrescens 204 205 [16], Thermococcus fumicolans [17], Thermococcus sp. NA1 [18], Thermococcus 206 207 208 peptonophilus [19], Thermococcus zilligii [20], Thermococcus aggregans [21], 209 210 Thermococcus celer [22], Thermococcus marinus [23], Thermococcus pacificus [24], 211 212 Thermococcus thioreducens [25], Thermococcuswaiotapuensis [26], and 213 214 215 Thermococcus barophilus Ch5 [27]. All these DNA pols have been proven to be used 216 217 efficiently in routine PCR. 218 219 220 The euryarchaeon Thermococcus gammatolerans was isolated from a 221 222 hydrothermal vent located in the Gulf of California [28]. This euryarchaeon is a 223 224 hyperthermophilic archaeon with an optimal growth temperature of 88°C which can 225 226 227 fully withstand a 5.0 kGy dose of gamma irradiation without loss of viability [29], and 228 229 thus is the most radioresistant archaeon to date. In reason of its extreme tolerance of 230 231 232 extreme temperatures and irradiation, it was very interesting to test whether the DNA 233 234 4 235 236 237 238 239 240 polymerases of T. gammatolerans has any specific features in comparison to other 241 242 243 DNA pols from this family. The genome of T. gammatolerans encodes two DNA pols, 244 245 a Family B DNA pol (Tga PolB) and Family D DNA pol [29]. In this work, we 246 247 cloned the Tga PolB gene, expressed its product and characterized its biochemical 248 249 250 characteristics. The recombinant Tga PolB was found capable of bypassing uracil in a 251 252 template DNA, which is distinct from other Family B archaeal DNA pols. 253 254 255 Furthermore, biochemical characterization of Tga PolB is discussed. 256 257 Materials and methods 258 259 2.1. Materials 260 261 262 Materials were purchased from the following companies: pET-30a (+), Novagen 263 264 (Merck, Darmstadt, Germany); Plasmid Extraction Kit, PCR Cycle Pure Kit, and Gel 265 266 267 Extraction Kit, Omega (Guangzhou, China); Escherichia coliDH5α cells, Transgene 268 269 (Beijing, China); dNTPs, T4 DNA ligase, NdeI, XhoI, and Pfu DNA pol, Thermo 270 271 Fisher Scientific (Waltham, MA); Chemicals, Amresco (WA, USA). 272 273 274 2.2. DNA substrates 275 276 All the oligonucleotides used were synthesized by Sangon Company (Shanghai, 277 278 279 China) and their sequences are listed in Table 1. Primed DNA substrates were 280 281 prepared by annealing the Cy3-labeled oligonucleotide with its complementary 282 283 oligonucleotides in an annealing buffer containing 20 mM Tris-Cl pH 8.0 and 100 284 285 o 286 mM NaCl. The annealing reactions were performed at 100 C for 5 min and cooled 287 288 slowly to room temperature at least 4 hours. 289 290 291 2.3. Cloning of the Tga PolB encoding gene 292 293 5 294 295 296 297 298 299 The TGAM_RS07365 gene encoding a Family BDNA pol of T. gammatolerans 300 301 302 was amplified by Pfu DNA pol using the genomic DNA as the template in the 303 304 presence of the forward primer (5′-GGG AAT TCC ATA TGC 305 306 ATTCTCGATACCGACTACATC-3′, the italic nucleotides represent NdeI restriction 307 308 309 site) and the reverse primer (5′- CCG CTC GAG TCA CTT CTT CCC TTT CAC 310 311 CTT CAG-3′, the italic nucleotides represent XhoI restriction site).The PCR product 312 313 314 was extracted and cleaved by NdeI and XhoI, and cloned into a pET-30a (+) vector.