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Plant and Cell Physiology Advance Access originally published online on October 6, 2009
Plant and Cell Physiology 2009 50(11):1965-1976; doi:10.1093/pcp/pcp140
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Arabidopsis Replication Protein A 70a is Required for DNA Damage Response and Telomere Length Homeostasis

Yoshiyuki Takashi, Yuki Kobayashi, Kan Tanaka and Katsunori Tamura*

Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan

*Corresponding author: E-mail, ktamura{at}iam.u-tokyo.ac.jp; Fax, +81-3-5841-8476.


  Abstract

Replication protein A1 (RPA1/RPA70) forms a heterotrimeric complex together with RPA2/RPA32 and RPA3/RPA14 subunits which plays essential roles in various aspects of DNA metabolism including replication, repair, recombination and telomere maintenance. Compared with RPA70 in yeast and mammals, limited information is available about the factor in plants. In this study, we analyzed the functions of AtRPA70a, which is most similar to human RPA70 among four paralogs in Arabidopsis thaliana. RNA blot analysis showed that AtRPA70a is expressed ubiquitously in plant organs containing differentiated and meristematic tissues, while its expression was up-regulated in response to DNA damage stress. Yeast two-hybrid and co-immunoprecipitation analyses showed that AtRPA70a interacted preferentially with Arabidopsis RPA32a, one of two paralogs. Inactivation of AtRPA70a by T-DNA insertion did not affect growth under normal conditions, but resulted in increased sensitivity to genotoxic agents such as methylmethane sulfonate, bleomycin and hydroxyurea. Terminal restriction fragment analysis revealed that telomere lengths in an AtRPA70a-deficient line were significantly larger than in the wild type, whereas those in the mutant expressing antisense AtTERT (telomerase catalytic subunit gene) were shortened during successive generations. These results demonstrate that AtRPA70a is involved in repair of double-strand DNA breaks and possibly contributes to telomerase-dependent telomere length regulation.

Keywords: Arabidopsis thaliana - Cell cycle checkpoint - DNA repair - Telomere maintenance

Abbreviations: AD, activation domain; 3-AT, 3-amino-1',2',3'-triazole; ATRIP, ATR-interacting protein; BD, binding domain; CaMV, cauliflower mosaic virus; DIG, digoxigenin; DSB, double-strand break; HA, hemagglutinin; HR, homologous recombination; HU, hydroxyurea; MEGA, Molecular Evolutionary Genetics Analysis; MMS, methylmethane sulfonate; MS, Murashige–Skoog; NHEJ, non-homologous end joining; OB-fold, oligonucleotide-binding fold; ONPG, o-nitrophenyl-β-D-galactopyranoside; RFA, replication factor A; RPA, replication protein A; RT–PCR, reverse transcription–PCR; ssDNA, single-stranded DNA; TRF, terminal restriction fragment; UTR, untranslated region; Y2H, yeast two-hybrid.

(Received June 16, 2009; Accepted September 30, 2009)
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