Abstract
Flap endonuclease-1 (FEN-1) is an important enzyme involved in DNA replication and repair. We isolated a 1.4 kb cDNA from rice (Oryza sativa), termed OsFEN-1, encoding a protein which shows homology with the eukaryotic FEN-1 proteins. OsFEN-1 protein was overexpressed in Escherichia coli and purified to near homogeneity. DNA cleavage analysis using different branched DNA structures indicated that OsFEN-1 protein possesses both 5′-flap endonuclease and 5′ to 3′ double-stranded DNA exonuclease activities. OsFEN-1 protein incises a 5′-flap and 5′-pseudo Y structure one base 3′ of the branched point in the duplex region. The enzymatic properties indicated that we succeeded in obtaining the gene and the protein of a plant counterpart of FEN-1. OsFEN-1 transcripts were expressed strongly in proliferating tissues such as root tips and young leaves that contain root apical meristem and marginal meristem, respectively. No expression was detected in mature leaves although the leaves were exposed to UV. We analyzed the spatial distribution pattern of OsFEN-1 transcripts by in situ hybridization. In the shoot apex, OsFEN-1 mRNA was abundant in the shoot apical meristem, tiller bud, leaf primordia, ligule primordia and marginal meristem of young leaves. In the roots, the transcript accumulated to high levels in the root apical meristem. Our results indicate that OsFEN-1 is expressed in tissues rich in proliferating cells, and its expression may be required for cell growth and organ formation.
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Alleva, J.L. and Doetsch, P.W. 1998. Characterization of Schizosaccharomyces pombe Rad2 protein, a FEN-1 homolog. Nucl. Acids Res. 26: 3645–3650.
Baba, A., Hasegawa, S. and Syono, K. 1986. Cultivation of rice protoplasts and their transformation mediated by Agrobacterium spheroplasts. Plant Cell 27: 463–471.
Barnes, C.J., Wahl, A.F., Shen, B., Park, M.S. and Bambara, R.A. 1996. Mechanism of tracking and cleavage of adduct-damaged DNA substrates by the mammalian 5'-to 3'-exonuclease/endonuclease RAD2 homologue 1 or flap endonuclease 1. J. Biol. Chem. 271: 29624–29631.
Batschauer, A. 1993. A plant gene for photolyase: an enzyme catalyzing the repair of UV-light-induced DNA damage. Plant J. 4: 705–709.
Bibikoba, M., Wu, B., Chi, E., Kim, K.H., Trautman, J.K. and Carroll, D. 1998. Characterization of FEN-1 from Xenopus laevis. cDNA cloning and role in DNA metabolism. J. Biol. Chem. 273: 34222–34229.
Britt, A.B. 1996. DNA damage and repair in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 75–100.
Britt, A.B., Chen, J.J., Wykoff, D. and Mitchell, D. 1993. A UV-sensitive mutant of Arabidopsis defective in the repair of pyrimidine-pyrimidinone (6-4) dimers. Science 261: 1571–1574.
Culligan, K.M. and Hays, J.B. 1997. DNA mismatch repair in plants. Plant Physiol. 115: 833–839.
Freudenreich, C.H., Kantrow, S.M. and Zakian, V.A. 1998. Expansion and length-dependent fragility of CTG repeats in yeast. Science 279: 853–856.
Hata, S., Kouchi, H., Tanaka, Y., Minami, E., Matsumoto, T., Suzuka, I. and Hashimoto, J. 1992. Identification of carrot cDNA clones encoding a second putative proliferating cell-nuclear antigen, DNA polymerase δ auxiliary protein. Eur. J. Biochem. 203: 367–371.
Harrington, J.J. and Lieber, M.R. 1994. The characterization of a mammalian DNA structure-specific endonuclease. EMBO J. 13: 1235–1246.
Harrington, J.J. and Lieber, M.R. 1995. DNA structural elements required for FEN-1 binding. J. Biol. Chem. 270: 4503–4508.
Hiraoka, L.R., Harrington, J.J., Gerhard, D.S., Lieber, M.R. and Hsieh, C.L. 1995. Sequence of human FEN-1, a structurespecific endonuclease, and chromosomal localization of the gene (FEN1) in mouse and human. Genomics 25: 220–225.
Hosfield, D.J., Mol, C.D., Shen, B. and Tainer, J.T. 1998. Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity. Cell 95: 135–146.
Ishimi, Y., Claude, A., Bullock, P. and Hurwitz, J. 1988. Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J. Biol. Chem. 263: 19723–19733.
Johnson, R.E., Gopala, K.K., Prakash, L. and Prakash, S. 1995. Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA. Science 269: 238–240.
Kimura, S., Kai, M., Kobayashi, H., Suzuki, A., Morioka, H., Otsuka, E. and Sakaguchi, K. 1997. A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence. Nucl. Acids Res. 25: 4970–4976.
Kimura, S., Takenouchi, M., Hatanaka, M., Seto, H., Kouroku, Y. and Sakaguchi, K. 1998. An ATP-inhibited endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence. Planta 206: 641–648.
Klungland, A. and Lindahl, T. 1997. Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1). EMBO J. 16: 3341–3348.
Kuriyan, J. and O'Donnell, M. 1993. Sliding clamps of DNA polymerases. J. Mol. Biol. 234: 915–925.
Landry, L.G., Stapleton, A.E., Jim, J., Hoffman, P., Hays, J.B., Walbot, V. and Last, R.L. 1997. An Arabidopsis photolyase mutant is hypersensitive to ultraviolet-B radiation. Proc. Natl. Acad. Sci. USA 94: 328–332.
Li, J., Ou-Lee, T.-M., Raba, R., Amundson, R.G. and Last, R.L. 1993. Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation. Plant Cell 5: 171–179.
Li, X., Li, J., Harrington, J.J., Lieber, M.R. and Burgers, P.M.J. 1995. Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen. J. Biol. Chem. 270: 22109–22112.
Lieber, M.R. 1997. The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair. BioEssays 19: 233–240.
McLennan, A.G. 1987. DNA damage, repair, and mutagenesis. In: J.A. Bryant and V.L. Dunham (Eds.), DNA Replication in Plants, CRC Press, Boca Raton, FL, pp. 135–186.
Matsumoto, Y., Kim, K. and Bogenhagen, D.F. 1994. Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair. Mol. Cell Biol. 14: 6187–6197.
Murray, J.M., Tavassoli, M., Al-Harithy, R., Sheldrick, K.S., Lehmann, A.R., Carr, A.M. and Watts, F.Z. 1994. Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage. Mol. Cell Biol. 14: 4878–4888.
Reagan, M.S., Pittenger, C., Siede, W. and Friedberg, E.C. 1995. Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene. J. Bact. 177: 364–371.
Robins, P., Pappin, D.J.C., Wood, R.D. and Lindahl, T. 1994. Structural and functional homology between mammalian DNase IV and the 5'-nuclease domain of Escherichia coli DNA polymerase I. J. Biol. Chem. 269: 28535–28538.
Rumbaugh, J.A., Murante, R.S., Shi, S. and Bambara, R.A. 1997. Creation and removal of embedded ribonucleotides in chromosomal DNA during mammalian Okazaki fragment processing. J. Biol. Chem. 272: 22591–22599.
Sato, Y., Hong, S.-K., Tagiri, A., Kitano, H., Yamamoto, N., Nagato, Y. and Matsuoka, M. 1996. A rice homeobox gene, OSH1, is expressed before organ differentiation in a specific region during early embryogenesis. Proc. Natl. Acad. Sci. USA 93: 8117–8122.
Seto, H., Hatanaka, M., Kimura, S., Oshige, M., Tsuya, Y., Mizushina, Y., Sawado, T., Aoyagi, N., Matsumoto, T., Hashimoto, J. and Sakaguchi, K. 1998. Purification and characterization of a 100 kDa DNA polymerase from cauliflower inflorescence. Biochem. J. 332: 557–563.
Shen, B., Nolan, J.P., Sklar, L.A. and Park, M.S. 1997. Functional analysis of point mutations in human flap endonuclease-1 active site. Nucl. Acids Res. 25: 3332–2228.
Shivji, M.K.K., Kenny, M.K. and Wood, R.D. 1992. Proliferating cell nuclear antigen is required for DNA excision repair. Cell 69: 367–374.
Smith, M.L., Chen, I.T., Zhan, Q., Bae, I., Chen, C.Y., Gilmer, T.M., Kastan, M.B., O'Connor, P.M. and Fornace, A.J. Jr. 1994. Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science 266: 1376–1380.
Sommers, C.H., Miller, E.J., Dujon, B., Prakash, S. and Prakash, L. 1995. Conditional lethality of null mutations in RTH1 that encodes the yeast counterpart of a mammalian 5'-to 3'-exonuclease required for lagging strand DNA synthesis in reconstituted systems. J. Biol. Chem. 370: 4193–4196.
Stapleton, A.E. and Walbot, V. 1994. Flavonoids protect maize DNA from the induction of ultraviolet radiation damage. Plant Physiol. 105: 881–889.
Stapleton, A.E., Thornber, C.S. and Walbot, V. 1997. UV-B component of sunlight causes measurable damage in field grown maize (Zea mays L.): developmental and cellular heterogeneity of damage and repair. Plant Cell Envir 20: 279–290.
Suzuka, I., Hata, S., Matsuoka, M., Kosugi, S. and Hashimoto, J. 1991. Highly conserved structure of proliferating cell nuclear antigen (DNA polymerase δ auxiliary protein) gene in plants. Eur. J. Biochem. 195: 571–575.
Waga, S., Hannon, G.J., Beach, D. and Stillman, B. 1994a. The p21 inhibitor of cyclin-dependent kinases controls DNA replication by interaction with PCNA. Nature 369: 574–578.
Waga, S., Bauer, G. and Stillman, B. 1994b. Reconstitution of complete SV40 DNA replication with purified replication factors. J. Biol. Chem. 269: 10923–10934.
Walbot, V. and Cullis, C.A. 1985. Rapid genomic change in plants. Annu. Rev. Plant Physiol. 36: 367–396.
Warbrick, E., Lane, D.P., Glover, D.M. and Cox, L.S. 1997. Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair. Oncogene 14: 2313–2321.
Warbrick, E., Lane, D.P., Glover, D.M. and Cox, L.S. 1995. A small peptide inhibitor of DNA replication defines the site of interaction between the cyclin-dependent kinase inhibitor p21WAF1 and proliferating cell nuclear antigen. Curr. Biol. 5: 275–282.
Waseem, N.H., Labib, K., Nurse, P. and Lane, D.P. 1992. Isolation and analysis of the fission yeast gene encoding polymerase delta accessory protein PCNA. EMBO J. 11: 5111–5120.
Wu, X., Li, J., Li, X., Hsieh, C.L., Burgers, P.M. and Lieber, M.R. 1996. Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA. Nucl. Acids Res. 24: 2036–2043.
Xiong, Y., Zhang, H. and Beach, D. 1992. D type cyclins associate with multiple protein kinases and the DNA replication and repair factor PCNA. Cell 71: 505–514.
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Kimura, S., Ueda, T., Hatanaka, M. et al. Plant homologue of flap endonuclease-1: molecular cloning, characterization, and evidence of expression in meristematic tissues. Plant Mol Biol 42, 415–427 (2000). https://doi.org/10.1023/A:1006349511964
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DOI: https://doi.org/10.1023/A:1006349511964