Regulation of mammalian replication origin usage in Xenopus egg extract.
Xenopus embryos initiate replication at random closely spaced sites until a certain concentration of nuclei is achieved within the embryo, after which fewer, more specific chromosomal sites are utilized as origins. We have examined the relationship between nucleo-cytosolic ratio and origin specification when Chinese hamster ovary (CHO) cell nuclei are introduced into Xenopus egg extracts. At concentrations of intact late-G1-phase nuclei that approximate early Xenopus embryos, the entire genome was duplicated nearly 4 times faster than in culture, accompanied by a de-localization of initiation sites at the dihydrofolate reductase (DHFR) locus. As the concentration of nuclei was increased, the number of initiation sites per nucleus decreased and initiation at the DHFR locus became localized to the physiologically utilized DHFR origin. Origin specification was optimal at nuclear concentrations that approximate the Xenopus mid-blastula transition (MBT). Higher concentrations resulted in an overall inhibition of DNA synthesis. By contrast, with intact early G1-phase nuclei, replication initiated at apparently random sites at all concentrations, despite an identical relationship between nucleo-cytosolic ratio and replicon size. Furthermore, permeabilization of late-G1-phase nuclei, using newly defined conditions that preserve the overall rate of replication, eliminated site-specificity, even at nuclear concentrations optimal for DHFR origin recognition. These data show that both nucleo-cytosolic ratio and nuclear structure play important but independent roles in the regulation of replication origin usage. Nucleo-cytosolic ratio clearly influences the number of replication origins selected. However, titration of cytosolic factors is not sufficient to focus initiation to specific sites. An independent mechanism, effecting changes within G1-phase nuclei, dictates which of many potential initiation sites will function as an origin.
Dimitrova
D S
DS
Department of Biochemistry and Molecular Biology, SUNY Health Science Center, Syracuse, NY 13210, USA.
Gilbert
D M
DM
D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication; D005260:Female; D016193:G1 Phase; D066298:In Vitro Techniques; D009685:Nuclear Envelope / Q000378:metabolism; D010063:Ovum / Q000378:metabolism*; D018741:Replication Origin; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014981:Xenopus
D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase