Abstract
Small plasmids replicate efficiently in unfertilized Xenopus eggs provided they are injected before rather than after activation of the cell cycle. Here we use Xenopus egg extracts to test the hypothesis that efficient replication results from chromatin assembly prior to activation giving preloaded plasmids a head start toward the formation of a replicating pseudonucleus (Sanchez, J.A., Marek, D., and Wangh, L.J. (1992) J. Cell Sci. 103, 907-918). As in ovum, plasmid DNA preincubated in unactivated egg cytoplasmcytostatic factor extracts) replicate more efficiently after extract activation than does the same DNA added to the same extract after activation. Unlike in ovum, however, plasmids that replicate efficiently in vitro do not assemble into chromatin during preincubation and become topologically knotted instead. But even DNA knotting does not explain subsequent efficient replication. Also, plasmids preassembled into chromatin in vitro do not replicate efficiently in activated egg cytoplasm unless first preincubated in a CSF extract. We conclude that unactivated eggs contain replication-enhancing activities that can act independently of plasmid chromatin assembly and DNA topology. These postulated "preloading" factor(s) may be related to licensing factor, an activity that controls initiation of DNA replication in eukaryotic cells. The experimental conditions described here will permit characterization of preloading/licensing factor(s) in the context of a small plasmid substrate.
Highlights
Our previous studies pointed to early chromatin assembly as an important step leading to plasmid replication in intact eggs
We have suggested that chromatin assembly before the start of the cell cycle leads to efficient replication after activation because it gives molecules a head start toward the formation of pseudonuclei
This paper describes a new experimental system for the efficient replication of small circular plasmid DNAs in extracts prepared from unfertilized Xenopus eggs
Summary
Our previous studies pointed to early chromatin assembly as an important step leading to plasmid replication in intact eggs. These investigators argue that eukaryotic nuclei normally replicate only once per cell cycle because they have to pass through mitosis, or at least experience nuclear envelope breakdown, before they can initiate DNA synthesis a second time Given these alternative possibilities, we decided to directly determine whether prior chromatin assembly accounts for efficient plasmid replication after the start of the cell cycle. In the course of this investigation, we discovered that CSF extracts cause closed circular plasmid molecules to become topologically knotted This observation led us to examine whether DNA knotting in unactivated egg cytoplasm might account for efficient in vitro replication. The in vitro system described here will permit characterization of this and other replication-enhancing activities of the mitotic egg cytoplasm in the context of an characterized plasmid substrate instead of the complex genome of whole eukaryotic nuclei
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