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¹ Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, NY 13210
² Department of Opthamology, State University of New York Upstate Medical University, Syracuse, NY 13210

Address correspondence to David M. Gilbert, Dept. of Biochemistry and Molecular Biology, 750 East Adams St., S.U.N.Y. Medical University, Syracuse, NY 13210. Tel.: (315) 464-8723. Fax: (315) 464-8750. E-mail: gilbertd{at}upstate.edu

We have examined the distribution of early replicating origins on stretched DNA fibers when nuclei from CHO cells synchronized at different times during G1 phase initiate DNA replication in Xenopus egg extracts. Origins were differentially labeled in vivo versus in vitro to allow a comparison of their relative positions and spacing. With nuclei isolated in the first hour of G1 phase, in vitro origins were distributed throughout a larger number of DNA fibers and did not coincide with in vivo origins. With nuclei isolated 1 h later, a similar total number of in vitro origins were clustered within a smaller number of DNA fibers but still did not coincide with in vivo origins. However, with nuclei isolated later in G1 phase, the positions of many in vitro origins coincided with in vivo origin sites without further change in origin number or density. These results highlight two distinct G1 steps that establish a spatial and temporal program for replication.

Key Words: DNA replication; origins of replication; replication timing; cell cycle; G1 phase

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