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

Xenopus egg extracts initiate replication at specific origin sites within m ammalian G1-phase nuclei. Similarly, S-phase extracts from Saccharomyces ce revisiae initiate DNA replication within yeast nuclei at specific yeast ori gin sequences. Here we show that Xenopus egg extracts can initiate DNA repl ication within G1-phase yeast nuclei but do not recognize yeast origin sequ ences. 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 s pecificity of initiation within the yeast 2 mum plasmid as well as in the v icinity of the chromosomal origin ARS1 was evaluated by neutral two-dimensi onal gel electrophoresis of replication intermediates. At both locations, r eplication was found to initiate outside of the ARS element. Manipulation o f both cis- and trans-acting elements in the yeast genome before introducti on of nuclei into Xenopus egg extract may provide a system with which to el ucidate the requirements for vertebrate origin recognition. (C) 2000 Wiley- Liss, Inc.