Cloning and Characterization of an Arabidopsis thaliana

cDNA and genomic clones encoding DNA topoisomerase I were isolated from Arabidopsis thaliana Xgtl1 and XFix libraries by low stringency hybridization with a Saccharomyces cerevisiae TOPi probe. The cDNA clones include a 2748-base pair open reading frame predicting an amino acid sequence that is highly homologous to sequences encoded by TOP1 from yeast and human sources. The sequence of the upstream genomic region reveals two putative TATA-like elements and a purine-rich region, but no other obvious controlling elements. Southern blot analysis shows that the gene is present as a single copy in the Arabidopsis genome. When expressed in a S. cerevisiae top1 mutant under the control of the GAL1 promoter, the gene complements the phenotype caused by loss of topoisomerase activity and directs the expression of a protein that cross-reacts with a human anti-topoisomerase I antibody. DNA topoisomerases are a class of enzymes that share the ability to alter the topological state of DNA. Type I topoisomerases change the linking number of DNA in steps of one by the transient breakage of a single strand of DNA, whereas type II enzymes alter the linking number in integrals of two by introducing double-strand breaks (6, 11, 29, 30). Genes for the type I enzyme have been cloned from bacteria (26), Saccharomyces cerevisiae (25), Schizosaccharomyces pombe (27), and human cells (8, 13). The bacterial and eukaryotic genes are not homologous. However, the yeast clone is able to complement a bacterial topoisomerase I-deficient mutant (4), and recently a novel topoisomerase I gene from yeast with homology to the bacterial gene also was described (28). Topoisomerases have been implicated in a number of processes affecting DNA, including transcription, replication, and recombination. The enzymes are thought to resolve the topological constraints imposed by the double-stranded, helical nature of DNA. For example, topoisomerase II is required for resolution of recombined chromosomes in yeast (23). Topoisomerase I is required for plasmid recombination in Escherichia coli (10) and for the pairing of covalently closed circular plasmids in vitro (7). 1 Supported by grant No. GM40725 from the National Institute of Health (E.R.S.), training grant No. GM 07287 from the National Institute of Health (J.J.K.), and a Lavoisier Fellowship from the Ministere des Affaires Etrangeres and the Soci&n Nationale Elf Aquitaine (A.F.T.). 2 Present address: Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018. How topoisomerases are involved in recombination is not yet clear. Yeast topl and top2 mutations show increased recombination specifically at rDNA3 repeats (5, 16) and not at other repeated sequences. A yeast mutation that gives hyperrecombination of repeated sequences, hprl, maps to a gene with homology to the carboxyl terminus of TOP1 (1). Also, a yeast gene (TOP3) with homology to bacterial type I topoisomerase was identified in a screen for mutants with elevated rates of recombination between co elements (28). These genes probably have overlapping functions because double mutants of topl with either a top3 or an hprl mutation have severely decreased growth rates, even though single topl deletions grow normally. The HPR1 and TOPIII genes seem to affect only intrachromosomal repeats, and it is not clear whether analogous genes are involved in interchromosomal homologous recombination. We are interested in genetic recombination in Arabidopsis thaliana and have begun to analyze individual components that may be involved. Elsewhere (14), we report the purification and characterization of a type I topoisomerase from the closely related crucifer Brassica oleracea var italica (broccoli). Here, we describe the cloning and characterization of a gene from A. thaliana that encodes a type I topoisomerase and that we have named TOP1. The gene is present as a single copy in the Arabidopsis genome. The cloned cDNA has an open reading frame of 2748 nucleotides, predicting a highly charged, basic protein with a mol wt of 104,000. The predicted protein sequence has 45% identity to that of the S. pombe gene. When expressed in a S. cerevisiae topl mutant, in which recombination at rDNA is specifically elevated (5), the clone depresses rDNA recombination, indicating that it complements the mutation, and directs the expression of a protein that cross-reacts to an antibody made to human topoisomerase I. MATERIALS AND METHODS Strains and Libraries Strains and plasmids used and their sources are listed in Table I. The XFix (Stratagene) Arabidopsis genomic library and the Xgt1l (31) poly(A)-primed cDNA library were kindly provided by H. Goodman. The random hexamer-primed Xgtll Arabidopsis cDNA library was kindly provided by G. Fink. The human antitopoisomerase I antibody (AFCDC9) was from the Centers for Disease Control (Atlanta, Georgia). 'Abbreviation: rDNA, ribosomal DNA. 1493 www.plantphysiol.org on December 31, 2017 Published by Downloaded from Copyright © 1992 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 99, 1992 Table I. Strains and Plasmids Used Strain or Relevant Source or Plasmid Characteristics Reference E. coli XL1 recA l lac endA I gyrA96 thi hsdR 17 supE44 relA 1; F' proAB+ /ac/Q lacZAM15 Tn Io Y1090 A/acUl69 proA+ A/on araD139 strA supF [trpC22::Tn 10] hsdRhsdM+ mcrApMC9 (for the growth of the XgtlO and Xgtl 1 libraries) ER1458 Y1090, mcrB(for the growth of the XFix library) strains XTop104 XFix containing a 11.0-kb TOP1 genomic fragment in the Xhol site XTopl 15 XFix containing a 14.5-kb TOP1 genomic fragment in the Xhol site east CY184 MATa ade2-4 ura3-1 his3-1 1, 15 trpl-l leu2-3, 112 canl-100 rDNA::ADE2 CY185 CY184, top 1-7::LEU2 YAB100 CY184, pTop17O YAB101 CY184, pCP125 YAB102 YAB100 screened for URAphenotype lasmids PCB12 pYCp5O with a 3.8 kb HindlIl insert containing the yeast TOPI gene pKSColEl; Apr (for sequencing and subcloning) pTop 140 pKSwith 1.5-kb Arabidopsis genomic TOPI fragment pTop141 pKSwith 7.5-kb Arabidopsis genomic TOPI fragment pTop142 pKSwith 8.5-kb Arabidopsis genomic TOPI fragment pTop150 Subclone of cDNA insert from Arabidopsis TOP1 from poly(A)-primed library in pKSpTop152 Subclone of cDNA insert from Arabidopsis TOPI from poly(A)-primed library in pKSpTop160 pKSwith a complete copy of the Arabidopsis TOP1 cDNA in the Sacl site pTop170 pCP125 with a complete copy of the Arabidopsis TOP1 cDNA in the BamHl site pCP125 pYIP5 with CEN-ARS1 and GAL1-GAL1O Stratagene