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Accurate DNA synthesis by Sulfolobus solfataricus DNA polymerase B1 at high temperature

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Abstract

The accuracy of DNA synthesis by DNA polymerase B1 from the hyperthermophilic archaeon Sulfolobus solfataricus (Sso pol B1) at near the physiological temperature was investigated using M13-based mutational assays. Sso pol B1 showed replication fidelity similar to or higher than most viral, bacterial, and eukaryotic replicases. The fidelity of the enzyme was about three times as high at 70°C as at 55°C. Approximately two-thirds of the errors made by the enzyme were single-base substitutions, of which 58% were C → T transition. Frameshift mutations, mostly resulting from single-base deletions, accounted for 19% of the total errors. An exonuclease-deficient mutant of Sso pol B1 was three times as mutagenic as the wild-type enzyme, suggesting that the intrinsic proofreading function contributed only modestly to the fidelity of the enzyme. Kinetic assays showed that the frequencies of all possible misincorporations by an exonuclease-deficient triple-point mutant of Sso pol B1 ranged from 5.4 × 10−5 to 4.6 × 10−4. The high fidelity of this enzyme in DNA synthesis was based primarily on K m difference rather than V max difference. These properties of Sso pol B1 are consistent with the proposed role of the enzyme as a replicase in S. solfataricus.

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Acknowledgments

We are grateful to Dr. Thomas A. Kunkel, NIEH, NIH, for donating E. coli strains and bacteriophage M13mp2 and to Dr. Zucai Suo, Ohio State University, USA, for providing the expression vector for exonuclease-deficient triple-point mutant of Sso pol B1. This study was supported by grants from the National Natural Science Foundation of China (30730003 and 30621005); National Basic Research Program of China (2004CB719603 to L.H.) and the Chinese Academy of Sciences (KSCX2-YW-G-023).

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  1. Huiqiang Lou

    Present address: State Key Laboratories for AgroBiotechnology, China Agricultural University, No. 2 Yuanmingyuan Xilu, Haidian District, Beijing, 100193, China

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  1. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101, China

    Likui Zhang, Huiqiang Lou, Li Guo, Zhengyan Zhan, Zhenhong Duan, Xin Guo & Li Huang

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Correspondence to Li Huang.

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Communicated by A. Driessen.

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Zhang, L., Lou, H., Guo, L. et al. Accurate DNA synthesis by Sulfolobus solfataricus DNA polymerase B1 at high temperature. Extremophiles 14, 107–117 (2010). https://doi.org/10.1007/s00792-009-0292-9

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