CLONING, EXPRESSION AND CHARACTERIZATION OF A GENE WHICH ENCODES A TOPOISOMERASE-I WITH POSITIVE SUPERCOILING ACTIVITY IN PEA

We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on the conserved amino acid sequenc es of five eukaryotic topoisomerase I, a 386 bp fragment was PCR ampli fied 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 re maining portion of the gene. The total size of the gene was 3055 bp wi th an open reading frame of 2676 bp. The deduced structure of pea topo isomerase 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 am ino acid sequences of the pea topo I with the other eukaryotic topoiso merases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli system and the recombinant topois omerase purified to homogeneity. The purified protein relaxes both pos itive and negative supercoiled DNA in the absence of divalent cation M g2+. In the presence of Mg2+ ions the purified enzyme introduces posit ive supercoils a unique property not reported in any other organism ex cept in archaebacterial topoisomerase I. Polyclonal antibodies were ra ised against recombinant topoisomerase I and western blotting with sub -cellular fractions indicated the localization of this topoisomerase i n pea nuclei.