Initiation of DNA replication in Saccharomyces cerevisiae G1-phase nuclei by Xenopus egg extract.
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.
Wu
J R
JR
Shanghai Institute of Biochemistry and Shanghai Research Center of Life Science, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Wang
J W
JW
Gilbert
D M
DM
D000818:Animals; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism*; D004261:DNA Replication; D015180:Electrophoresis, Gel, Two-Dimensional; D016193:G1 Phase; D009865:Oocytes; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D014981:Xenopus
D002457:Cell Extracts