Cloning and sequencing of Schizosaccharomyces pombe DNA topoisomerase I gene, and effect of gene disruption

Abstract

We cloned the structural gene top1+ for Schizosaccharomyces pombe DNA topoisomerase I (topo I) by hybridization. An eight-fold increase of topo I relaxing activity was obtained in S. pombe cells transformed with multicopy plasmid with top1+ insert. Nucleotide sequence determination showed a hypothetical coding frame interrupted by two short introns, encoding a 812 residue polypeptide (M.W. 94,000), 43 residues longer than and 47% homologous to Saccharomyces cerevisiae topo I. We show that the top1 (null) strain made by gene disruption is viable, although its generation time is 20% longer than that of wild type. The top1 locus is mapped in the long arm of chromosome II, using the Leu+ marker integrated with the cloned top1+ sequence. We constructed a double mutant top1 (null) top2 (ts) and found its defective phenotype similar to that of previously obtained top1 (heat sensitive) top2 (ts). The other double mutant top1 (null) top2 (cs), however, was lethal. Our results suggest that top1+ gene of S. pombe is dispensable only if topo II activity is abundant. © 1987 IRL Press Limited.