APHIDICOLIN-SENSITIVE DNA-POLYMERASE IS INCORPORATED INTO THE CHROMATIN DURING NUCLEAR-ENVELOPE ASSEMBLY IN XENOPUS EGG EXTRACT

The mechanism for incorporation of aphidicolin-sensitive DNA polymeras e into reconstituting sperm nuclei was studied in a Xenopus egg extrac t cell-free system. Aphidicolin-sensitive DNA polymerase activity was sedimented along with the light membrane fraction of Xenopus egg extra ct on a discontinuous sucrose gradient. Treatment of the egg extract w ith Triton X-100 caused DNA polymerase activity to migrate to a lighte r density position at which free proteins were distributed. DNA polyme rase activity was incorporated into the reconstituting sperm nuclei fr om the egg extract, but no nuclear incorporation was observed in nucle i incubated in egg extracts which had been treated with Triton X-100 o r sonicated. The incorporation was also prohibited by several differen t treatments of the egg extract resulting in incomplete assembly of th e nuclear membrane on the sperm nuclei. On the other hand, there was n o inhibition of nuclear incorporation into the sperm nuclei reconstitu ting in the extracts which had been depleted of WGA-binding pore compl ex proteins or which contained a specific inhibitor of topoisomerase I I (ICRF-193). In these two cases, the nuclear double-layered membrane assembled normally, although in the former case the sperm nuclei lacke d lamina and did not initiate DNA replication, and in the latter case the sperm nuclei did not decondense but initiated DNA replication. Thu s, it is concluded that DNA polymerase activity is incorporated into t he reconstituting nuclei via the membraneous/particulate fraction of t he egg extract simultaneously with nuclear double-layered membrane ass embly. The lamina assembly and the transport system via the nuclear en velope pore complex are suggested not to participate in DNA polymerase nuclear incorporation. (C) 1995 Academic Press, Inc.