DNA Topoisomerase VI Is Essential for Endoreduplication in Arabidopsis

Endoreduplication is a common process in eukaryotes that involves DNA amplification without corresponding cell divisions. Cell size in various organisms has been linked to endoreduplication, but the molecular mechanisms are poorly understood. We have used a genetic strategy to identify molecules involved in endocycles in Arabidopsis. We isolated two extreme dwarf mutants, hypocotyl6 (hyp6) and root hairless2 (rhl2) [3], and cells of these mutants successfully complete only the first two rounds of endoreduplication and stall at 8C. In both mutants, large cell types, such as trichomes and some epidermal cells, that normally endoreduplicate their DNA are much reduced in size. We show that HYP6 encodes AtTOP6B, a plant homolog of the archaeal DNA topoisomerase VI subunit B, and that RHL2 encodes AtSPO11-3, one of the three Arabidopsis subunit A homologs. We propose that this topoisomerase VI complex is essential for the decatenation of replicated chromosomes during endocycles and that successive rounds of endoreduplication are required for the full growth of specific cell types.

[1]  J. Champoux.  DNA topoisomerases: structure, function, and mechanism. , 2001, Annual review of biochemistry.

[2]  E. Lam,et al.  Abundance of nuclear DNA topoisomerase II is correlated with proliferation in Arabidopsis thaliana. , 1994, Nucleic acids research.

[3]  Ken Shirasu,et al.  The RAR1 Interactor SGT1, an Essential Component of R Gene-Triggered Disease Resistance , 2002, Science.

[4]  M. Grelon,et al.  AtSPO11‐1 is necessary for efficient meiotic recombination in plants , 2001, The EMBO journal.

[5]  J. Parvin,et al.  DNA topoisomerase IIα is required for RNA polymerase II transcription on chromatin templates , 2001, Nature.

[6]  D. Galbraith,et al.  Systemic Endopolyploidy in Arabidopsis thaliana. , 1991, Plant physiology.

[7]  M. Hülskamp,et al.  Endoreduplication and development: rule without dividing? , 1998, Current opinion in plant biology.

[8]  E. Kondorosi,et al.  Plant cell-size control: growing by ploidy? , 2000, Current opinion in plant biology.

[9]  P. Forterre,et al.  Purification of a DNA topoisomerase II from the hyperthermophilic archaeon Sulfolobus shibatae. A thermostable enzyme with both bacterial and eucaryal features. , 1994, Journal of Biological Chemistry.

[10]  J. Wang,et al.  Reconstitution of DNA topoisomerase VI of the thermophilic archaeon Sulfolobus shibatae from subunits separately overexpressed in Escherichia coli. , 1998, Nucleic acids research.

[11]  A. Coleman,et al.  Relationship between Endopolyploidy and Cell Size in Epidermal Tissue of Arabidopsis. , 1993, The Plant cell.

[12]  M. Caboche,et al.  Cellular Basis of Hypocotyl Growth in Arabidopsis thaliana , 1997, Plant physiology.

[13]  H. Puchta,et al.  Molecular characterization of homologues of both subunits A (SPO11) and B of the archaebacterial topoisomerase 6 in plants. , 2001, Gene.

[14]  M. Caboche,et al.  Use of two-dimensional protein-pattern analysis for the characterization of Arabidopsis thaliana mutants , 1994, Planta.

[15]  H. Puchta,et al.  Molecular characterisation of two paralogous SPO11 homologues in Arabidopsis thaliana. , 2000, Nucleic acids research.

[16]  S. Keeney,et al.  Mechanism and control of meiotic recombination initiation. , 2001, Current topics in developmental biology.

[17]  F. Ausubel,et al.  A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. , 1993, The Plant journal : for cell and molecular biology.

[18]  T. Ohta,et al.  DNA Supercoiling Factor Localizes to Puffs on Polytene Chromosomes in Drosophila melanogaster , 1998, Molecular and Cellular Biology.

[19]  Martin Hülskamp,et al.  Genetic dissection of trichome cell development in Arabidopsis , 1994, Cell.

[20]  A. Schnittger,et al.  Pattern formation and cell differentiation: trichomes in Arabidopsis as a genetic model system. , 1999, International review of cytology.

[21]  L. Dolan,et al.  Structural and genetic analysis of epidermal cell differentiation in Arabidopsis primary roots. , 1997, Development.

[22]  J. Ecker,et al.  Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. , 1994, Genomics.

[23]  J. Berger,et al.  Structure and function of an archaeal topoisomerase VI subunit with homology to the meiotic recombination factor Spo11 , 1999, The EMBO journal.

[24]  J. Lebbink,et al.  DNA Topoisomerase VI Generates ATP-dependent Double-strand Breaks with Two-nucleotide Overhangs* , 2001, The Journal of Biological Chemistry.

[25]  A. Nicolas,et al.  An atypical topoisomerase II from archaea with implications for meiotic recombination , 1997, Nature.

[26]  JAMES C. Wang,et al.  Cellular roles of DNA topoisomerases: a molecular perspective , 2002, Nature Reviews Molecular Cell Biology.

[27]  J. Doonan,et al.  Plant‐adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans , 1998, Molecular microbiology.

[28]  R. Hellens,et al.  pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation , 2000, Plant Molecular Biology.