DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts.
Cell-free extracts of Xenopus eggs will replicate plasmid DNA molecules under normal cell cycle control. We have used the neutral/neutral 2-D gel technique to map the sites at which DNA replication initiates in this system. Three different plasmids were studied: one containing the Xenopus rDNA repeat, one containing single copy Xenopus genomic DNA, and another containing the yeast 2 microns replication origin. 2-D gel profiles show that many potential sites of initiation are present on each plasmid, and are randomly situated at the level of resolution of this technique (500-1000 bp). Despite the abundance of sites capable of supporting the initiation of replication, pulse-chase experiments suggest that only a single randomly situated initiation event occurs on each DNA molecule. Once initiation has taken place, conventional replication forks appear to move away from this site at a rate of about 10nt/second, similar to the rate observed in vivo.
Mahbubani
H M
HM
ICRF Clare Hall Laboratories, Potters Bar, Herts, UK.
Paull
T
T
Elder
J K
JK
Blow
J J
JJ
D000818:Animals; D002457:Cell Extracts / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D010063:Ovum / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D014982:Xenopus laevis
D002457:Cell Extracts; D004275:DNA, Ribosomal