J Cell Biochem 2000 Sep 18;801:73-84. doi: 10.1002/1097-4644(20010101)80:1<73::aid-jcb70>3.0.co;2-7.

Initiation of DNA replication in Saccharomyces cerevisiae G1-phase nuclei by Xenopus egg extract.

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Xenopus egg extracts initiate replication at specific origin sites within mammalian G1-phase nuclei. Similarly, S-phase extracts from Saccharomyces cerevisiae initiate DNA replication within yeast nuclei at specific yeast origin sequences. Here we show that Xenopus egg extracts can initiate DNA replication within G1-phase yeast nuclei but do not recognize yeast origin sequences. When G1-phase yeast nuclei were introduced into Xenopus egg extract, semiconservative, aphidicolin-sensitive DNA synthesis was induced after a brief lag period and was restricted to a single round of replication. The specificity of initiation within the yeast 2 microm plasmid as well as in the vicinity of the chromosomal origin ARS1 was evaluated by neutral two-dimensional gel electrophoresis of replication intermediates. At both locations, replication was found to initiate outside of the ARS element. Manipulation of both cis- and trans-acting elements in the yeast genome before introduction of nuclei into Xenopus egg extract may provide a system with which to elucidate the requirements for vertebrate origin recognition.

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