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Small-angle X-ray scattering analysis reveals the ATP-bound monomeric state of the ATPase domain from the homodimeric MutL endonuclease, a GHKL phosphotransferase superfamily protein

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Abstract

DNA mismatch repair is an excision system that removes mismatched bases chiefly generated by replication errors. In this system, MutL endonucleases direct the excision reaction to the error-containing strand of the duplex by specifically incising the newly synthesized strand. Both bacterial homodimeric and eukaryotic heterodimeric MutL proteins belong to the GHKL ATPase/kinase superfamily that comprises the N-terminal ATPase and C-terminal dimerization regions. Generally, the GHKL proteins show large ATPase cycle-dependent conformational changes, including dimerization-coupled ATP binding of the N-terminal domain. Interestingly, the ATPase domain of human PMS2, a subunit of the MutL heterodimer, binds ATP without dimerization. The monomeric ATP-bound state of the domain has been thought to be characteristic of heterodimeric GHKL proteins. In this study, we characterized the ATP-bound state of the ATPase domain from the Aquifex aeolicus MutL endonuclease, which is a homodimeric GHKL protein unlike the eukaryotic MutL. Gel filtration, dynamic light scattering, and small-angle X-ray scattering analyses clearly showed that the domain binds ATP in a monomeric form despite its homodimeric nature. This indicates that the uncoupling of dimerization and ATP binding is a common feature among bacterial and eukaryotic MutL endonucleases, which we suggest is closely related to the molecular mechanisms underlying mismatch repair.

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Abbreviations

MMR:

Mismatch repair

aqMutL:

Aquifex aeolicus MutL

NTD:

N-terminal domain

CTD:

C-terminal domain

DLS:

Dynamic light scattering

SAXS:

Small-angle X-ray scattering

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Acknowledgments

The authors thank Kayoko Matsumoto for the experiment involving overexpression of the proteins, Aimi Osaki for the assistance of protein purifications. Authors also thank Masao Inoue for his critical reading of this manuscript. The SAXS experiments in this study were performed at BL45XU in SPring-8 (Proposals 20110033 and 20120009). This work was supported by Management Expenses Grants from the government to RIKEN.

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Authors and Affiliations

  1. RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-Gun, Hyogo, 679-5148, Japan

    Hitoshi Iino, Takaaki Hikima & Masaki Yamamoto

  2. Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka, 560-0043, Japan

    Yuya Nishida & Seiki Kuramitsu

  3. Department of Biochemistry, Osaka Medical College, 2-7, Daigaku-machi, Takatsuki, Osaka, 569-8686, Japan

    Kenji Fukui

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  1. Hitoshi Iino
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  2. Takaaki Hikima
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  3. Yuya Nishida
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  4. Masaki Yamamoto
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  5. Seiki Kuramitsu
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Correspondence to Kenji Fukui.

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Communicated by F. Robb.

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Iino, H., Hikima, T., Nishida, Y. et al. Small-angle X-ray scattering analysis reveals the ATP-bound monomeric state of the ATPase domain from the homodimeric MutL endonuclease, a GHKL phosphotransferase superfamily protein. Extremophiles 19, 643–656 (2015). https://doi.org/10.1007/s00792-015-0745-2

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