A large circular minichromosome of Schizosaccharomyces pombe requires a high dose of type II DNA topoisomerase for its stabilization.
We have constructed circular minichromosomes, ranging in size from 36 to 110 kb, containing the centromeric repeats of Schizosaccharomyces pombe cen3. Comparison of their mitotic stability showed that the circular minichromosomes became more unstable with increasing in size, however, a linear cen3 minichromosome, which is almost the same size as the largest circular one tested, does not show such instability. High levels of expression of the top2+ (type II DNA topoisomerase; topo II) but not top1+ gene (type I DNA topoisomerase) suppressed the instability of the largest circular minichromosome, whereas partial inactivation of topo II dramatically destabilized the minichromosome. A mutant topo II, defective in nuclear localization but still retaining its in vitro relaxation activity, did not stabilize the circular minichromosome. These results indicate that endogenous type II DNA topoisomerase is insufficient for accurate segregation of the circular minichromosome. In addition, the replication of the minichromosomal DNA appears to proceed normally, because the presence of the unstable minichromosome did not cause G2 delay. A likely cause of the instability is intertwining of the minichromosome DNA possibly occurring after DNA replication. An interaction between topo II and the centromeric repeats is implied by the finding that multiple copies of the centromeric repeat, dg-dh, affect stability of the minichromosome similarly to top2+ gene dosage.
Murakami
S
S
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Yanagida
M
M
Niwa
O
O
D015825:Chromosomes, Fungal / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D016195:G2 Phase / Q000235:genetics; D008938:Mitosis / Q000235:genetics; D010641:Phenotype; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D012303:Ring Chromosomes; D012568:Schizosaccharomyces / Q000235:genetics*; D014170:Transformation, Genetic
D004250:DNA Topoisomerases, Type II