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Enzymatic Activities of RNase H Domains of HIV-1 Reverse Transcriptase with Substrate Binding Domains of Bacterial RNases H1 and H2

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Abstract

Thermotoga maritima RNase H1 and Bacillus stearothermophilus RNase H2 have an N-terminal substrate binding domain, termed hybrid binding domain (TmaHBD), and N-terminal domain (BstNTD), respectively. HIV-1 reverse transcriptase (RT) is a heterodimer consisting of a P66 subunit and a P51 subunit. The P66 subunit contains a C-terminal RNase H domain, which exhibits RNase H activity either in the presence of Mg2+ or Mn2+ ions. The isolated RNase H domain of HIV-1 RT (RNHHIV) is inactive, possibly due to the lack of a substrate binding ability, disorder of a loop containing His539, and increased flexibility. To examine whether the activity of RNHHIV is restored by the attachment of TmaHBD or BstNTD to its N-terminus, two chimeric proteins, TmaHBD-RNHHIV and BstNTD-RNHHIV, were constructed and characterized. Both chimeric proteins bound to RNA/DNA hybrid more strongly than RNHHIV and exhibited enzymatic activity in the presence of Mn2+ ions. They did not exhibit activity or exhibited very weak activity in the presence of Mg2+ ions. These results indicate that TmaHBD and BstNTD function as an RNA/DNA hybrid binding tag, and greatly increase the substrate binding affinity and Mn2+-dependent activity of RNHHIV but do not restore the Mg2+-dependent activity of RNHHIV.

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Acknowledgments

We thank Dr. Y. Koga and Dr. E. Kanaya for helpful discussions. This work was supported in part by the Grant (24380055) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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  1. Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Etin-Diah Permanasari, Kiyoshi Yasukawa & Shigenori Kanaya

  2. Division Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan

    Etin-Diah Permanasari, Kiyoshi Yasukawa & Shigenori Kanaya

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  1. Etin-Diah Permanasari
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Correspondence to Shigenori Kanaya.

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Permanasari, ED., Yasukawa, K. & Kanaya, S. Enzymatic Activities of RNase H Domains of HIV-1 Reverse Transcriptase with Substrate Binding Domains of Bacterial RNases H1 and H2. Mol Biotechnol 57, 526–538 (2015). https://doi.org/10.1007/s12033-015-9846-5

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