Plant Molecular Biology 37: 773–784, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
Cloning, expression and characterization of a gene which encodes a
topoisomerase I with positive supercoiling activity in pea
and Krishna K. Tewari
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067,
author for correspondence);
Department of Molecular Biology and Biochemistry, University of
California, Irvine, CA 92717, USA
Received 7 October 1997; accepted 2 February 1998
Key words: heterologous expression, introduction of positive supercoils in DNA, Pisum sativum, RACE-PCR,
relaxation of DNA supercoils
We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on
the conserved amino acid sequences of ﬁve eukaryotic topoisomerase I, a 386 bp fragment was PCR ampliﬁed
using pea cDNA as template. This fragment was used as a probe to screen a pea cDNA library. Two partial
cDNA clones were isolated which were truncated at the 5
end. RACE-PCR was employed to isolate the remaining
portion of the gene. The total size of the gene was 3055 bp with an open reading frame of 2676 bp. The deduced
structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an
estimated pI of 9.3. A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic
topoisomerases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli
system and the recombinant topoisomerase puriﬁed to homogeneity. The puriﬁed protein relaxes both positive
and negative supercoiled DNA in the absence of divalent cation Mg
. In the presence of Mg
ions the puriﬁed
topoisomerase I. Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with
sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei.
The intertwining of the two strands of the DNA helix
presents a number of topological problems which the
cell must overcome in order to replicate, express,
recombine and segregate its genetic information [24,
36, 54, 55, 56, 57, 58, 59]. Both prokaryotic and
eukaryotic cells contain a class of enzymes known
as topoisomerases to resolve the topological demands
of DNA. Topoisomerases are classiﬁed into two cat-
egories based on their reaction mechanisms. Type I
enzymes nick and seal one of the two strands of the
DNA substrate and change the linking number of the
supercoiled DNA by steps of one , while type
The nucleotide sequence data reported appears in the EMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the
accession number Y14558.
II enzymes cause breakage and reunion of both the
strands and change the linking number by steps of two
In contrast to bacterial, viral, animal and yeast
systems where the topoisomerases have been extens-
ively studied , there are relatively few reports on
plant topoisomerases. Type I topoisomerase activity
has been described in wheat germ [18, 19], carrot cells
[1, 4, 5], pea [10, 11, 12], cauliﬂower , broccoli
, Arabidopsis  and cultured tobacco cells .
Inchloroplasts,type I topoisomerasehas been reported
in cauliﬂower, pea and spinach [23, 32, 35, 42, 47].
A type II topoisomerase activity has been reported in
nuclei of cauliﬂower [21, 22] pea , Arabidopsis
 and the chloroplasts of pea  and wheat .
Inmanycases onlythe presenceofa topoisomerase
activity or its partial puriﬁcation has been reported [4,