Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase

doi:10.1083/jcb.117.5.921

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Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase

CE Shamu and AW Murray
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.

We have produced metaphase spindles and induced them to enter anaphase in vitro. Sperm nuclei were added to frog egg extracts, allowed to replicate their DNA, and driven into metaphase by the addition of cytoplasm containing active maturation promoting factor (MPF) and cytostatic factor (CSF), an activity that stabilizes MPF. Addition of calcium induces the inactivation of MPF, sister chromatid separation and anaphase chromosome movement. DNA topoisomerase II inhibitors prevent chromosome segregation at anaphase, demonstrating that the chromatids are catenated at metaphase and that decatenation occurs at the start of anaphase. Topoisomerase II activity towards exogenous substrates does not increase at the metaphase to anaphase transition, showing that chromosome separation at anaphase is not triggered by a bulk activation of topoisomerase II.
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