Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos.
Chromosome replication initiates without sequence specificity at average intervals of approximately 10 kb during the rapid cell cycles of early Xenopus embryos. If the distribution of origins were random, some inter-origin intervals would be too long to be fully replicated before the end of S phase. To investigate what ensures rapid completion of DNA replication, we have examined the replication intermediates of plasmids of various sizes (5.3-42.2 kbp) in Xenopus egg extracts by two-dimensional gel electrophoresis and electron microscopy. We confirm that replication initiates without sequence specificity on all plasmids. We demonstrate for the first time that multiple initiation events occur on large plasmids, but not on small (<10 kb) plasmids, at average intervals of approximately 10 kb. Origin interference may prevent multiple initiation events on small plasmids. Multiple initiation events are neither synchronous nor regularly spaced. Bubble density is higher on later than on earlier replication intermediates, showing that initiation frequency increases throughout S phase, speeding up replication of late intermediates. We suggest that potential origins are abundant and randomly distributed, but that the increase of initiation frequency during S phase, and possibly origin interference, regulate origin activation to ensure rapid completion of replication.
Lucas
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I
Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm, Paris Cedex 05, 75230, France.
Chevrier-Miller
M
M
Sogo
J M
JM
Hyrien
O
O
D000818:Animals; D002457:Cell Extracts; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D004261:DNA Replication / Q000235:genetics*; D015180:Electrophoresis, Gel, Two-Dimensional; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D008854:Microscopy, Electron; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D018741:Replication Origin / Q000235:genetics*; D016196:S Phase / Q000235:genetics; D014982:Xenopus laevis; D015053:Zygote / Q000166:cytology / Q000378:metabolism
D002457:Cell Extracts; D004247:DNA