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Rice exonuclease-1 homologue, OsEXO1, that interacts with DNA polymerase λ and RPA subunit proteins, is involved in cell proliferation

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Abstract

Exonuclease 1, a class III member of the RAD2 nuclease family, is a structure-specific nuclease involved in DNA metabolism (replication, repair and recombination). We have identified a homologue to Exonuclease-1 from rice (Oryza sativa L. cv. Nipponbare) and have designated it O. sativa Exonuclease-1 (OsEXO1). The open reading frame of OsEXO1 encodes a predicted product of 836 amino acid residues with a molecular weight of 92 kDa. Two highly conserved nuclease domains (XPG-N and XPG-I) are present in the N-terminal region of the protein. OsEXO1-sGFP fusion protein transiently overexpressed in the onion epidermal cells localized to the nucleus. The transcript of OsEXO1 is highly expressed in meristematic tissues and panicles. Inhibition of cell proliferation by removal of sucrose from the medium or by the addition of cell cycle inhibitors decreased OsEXO1 expression. Functional complementation assays using yeast RAD2 member null mutants demonstrates that OsEXO1 is able to substitute for ScEXO1 and ScRAD27 functions. Yeast two-hybrid analysis shows that OsEXO1 interacted with rice DNA polymerase λ (OsPol λ), the 70 kDa subunit b of rice replication protein A (OsRPA70b), and the 32 kDa subunit 1 of rice replication protein A (OsRPA32-1). Irradiation of UV-B induces OsEXO1 expression while hydrogen peroxide treatment represses it. These results suggest that OsEXO1 plays an important role in both cell proliferation and UV-damaged nuclear DNA repair pathway under dark conditions.

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Abbreviations

EXO1:

Exonuclease-1

FEN-1:

Flap endonuclease-1

Pol:

DNA polymerase

RACE:

Rapid amplification of cDNA ends

RPA:

Replication protein A

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Acknowledgements

This work was supported in part by Sasakawa Grants for Science Fellows (SGSF), Grants-in Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (Grants Nos. 15570042, 17310115 and 17657020 to HS). We thank Dr. N. Kasai for generating 3D models of protein structures. We also thank Dr. A. Britt and Mr. N. Heufner for useful discussion and critical reading of the manuscript.

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Author notes

  1. Tomoyuki Furukawa

    Present address: Section of Plant Biology, University of California, Davis, Davis, CA, 95616, USA

Authors and Affiliations

  1. Division of Plant Biotechnology, Tissue Engineering Research Center, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan

    Tomoyuki Furukawa & Hiroaki Shimada

  2. Department of Biological Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan

    Tomohiro Imamura & Hiroaki Shimada

  3. National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Ibaraki, 305-8604, Japan

    Hiroko K. Kitamoto

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  1. Tomoyuki Furukawa
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  2. Tomohiro Imamura
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  4. Hiroaki Shimada
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Corresponding author

Correspondence to Hiroaki Shimada.

Additional information

Each nucleotide sequence data reported appears in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the Accession No. AB179767 for OsEXO1.

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11103_2008_9288_MOESM1_ESM.tif

Supplementary Fig. 1 A modeled 3D structure for OsEXO1 protein. (Left) X-ray structure of HEX1 protein (amino acids 15–313). This structure was used as a template for modeling of OsEXO1. (Right) Modeled 3D structure for OsEXO1 protein (amino acids 17–258). Arrowheads indicate conserved aspartic acids in XPG domains. (TIF 45332 kb)

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Furukawa, T., Imamura, T., Kitamoto, H.K. et al. Rice exonuclease-1 homologue, OsEXO1, that interacts with DNA polymerase λ and RPA subunit proteins, is involved in cell proliferation. Plant Mol Biol 66, 519–531 (2008). https://doi.org/10.1007/s11103-008-9288-6

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