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Characterization of six recombinant human RNase H2 bearing Aicardi-Goutiéres syndrome causing mutations.
J Biochem 2019JB

Abstract

Mammalian RNase H2 is a heterotrimeric enzyme consisting of one catalytic subunit (A) and two accessory subunits (B and C). RNase H2 is involved in the removal of a single ribonucleotide embedded in genomic DNA and removal of RNA of RNA/DNA hybrids. In humans, mutation of the RNase H2 gene causes a severe neuroinflammatory disorder Aicardi-Goutières syndrome (AGS). Here, we examined the activity and stability of six recombinant human RNase H2 variants bearing one AGS-causing mutation, A-G37S (Gly37 in the A subunit is replaced with Ser), A-N212I, A-R291H, B-A177T, B-V185G, or C-R69W. The activity of A-G37S was 0.3-1% of that of the wild-type RNase H2 (WT), while those of other five variants were 51-120%. In circular dichroism measurement, the melting temperatures of variants were 50-53 °C, lower than that of WT (56 °C). These results suggested that G37S had decreased activity and stability than WT, while other five variants had decreased stability but retained activity. In gel filtration chromatography of the purified enzyme preparation, WT migrated as a heterotrimer, while A-R291H eluted in two separate peaks containing either the heterotrimer or only the A subunit, suggesting that some AGS-causing mutations affect the heterotrimer-forming stability of RNase H2.

Authors+Show Affiliations

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.Section on Formation of RNA, Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31529068

Citation

Nishimura, Takuto, et al. "Characterization of Six Recombinant Human RNase H2 Bearing Aicardi-Goutiéres Syndrome Causing Mutations." Journal of Biochemistry, 2019.
Nishimura T, Baba M, Ogawa S, et al. Characterization of six recombinant human RNase H2 bearing Aicardi-Goutiéres syndrome causing mutations. J Biochem. 2019.
Nishimura, T., Baba, M., Ogawa, S., Kojima, K., Takita, T., Crouch, R. J., & Yasukawa, K. (2019). Characterization of six recombinant human RNase H2 bearing Aicardi-Goutiéres syndrome causing mutations. Journal of Biochemistry, doi:10.1093/jb/mvz073.
Nishimura T, et al. Characterization of Six Recombinant Human RNase H2 Bearing Aicardi-Goutiéres Syndrome Causing Mutations. J Biochem. 2019 Sep 16; PubMed PMID: 31529068.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Characterization of six recombinant human RNase H2 bearing Aicardi-Goutiéres syndrome causing mutations. AU - Nishimura,Takuto, AU - Baba,Misato, AU - Ogawa,Saori, AU - Kojima,Kenji, AU - Takita,Teisuke, AU - Crouch,Robert J, AU - Yasukawa,Kiyoshi, Y1 - 2019/09/16/ PY - 2019/04/02/received PY - 2019/09/02/accepted PY - 2019/9/19/entrez KW - AGS KW - activity KW - human RNase H2 KW - mutation KW - stability JF - Journal of biochemistry JO - J. Biochem. N2 - Mammalian RNase H2 is a heterotrimeric enzyme consisting of one catalytic subunit (A) and two accessory subunits (B and C). RNase H2 is involved in the removal of a single ribonucleotide embedded in genomic DNA and removal of RNA of RNA/DNA hybrids. In humans, mutation of the RNase H2 gene causes a severe neuroinflammatory disorder Aicardi-Goutières syndrome (AGS). Here, we examined the activity and stability of six recombinant human RNase H2 variants bearing one AGS-causing mutation, A-G37S (Gly37 in the A subunit is replaced with Ser), A-N212I, A-R291H, B-A177T, B-V185G, or C-R69W. The activity of A-G37S was 0.3-1% of that of the wild-type RNase H2 (WT), while those of other five variants were 51-120%. In circular dichroism measurement, the melting temperatures of variants were 50-53 °C, lower than that of WT (56 °C). These results suggested that G37S had decreased activity and stability than WT, while other five variants had decreased stability but retained activity. In gel filtration chromatography of the purified enzyme preparation, WT migrated as a heterotrimer, while A-R291H eluted in two separate peaks containing either the heterotrimer or only the A subunit, suggesting that some AGS-causing mutations affect the heterotrimer-forming stability of RNase H2. SN - 1756-2651 UR - https://www.unboundmedicine.com/medline/citation/31529068/Characterization_of_six_recombinant_human_RNase_H2_bearing_Aicardi-Goutiéres_syndrome_causing_mutations L2 - https://academic.oup.com/jb/article-lookup/doi/10.1093/jb/mvz073 DB - PRIME DP - Unbound Medicine ER -