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PMID: 7760792 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

Molecular and cellular biology ·Vol. 15 ·No. 6 ·1995-06-00 ·Pages 2942-54

Gilbert DM, Miyazawa H, DePamphilis ML

Abstract

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.

MeSH Terms
Animals CHO Cells/metabolism Cell Nucleus/metabolism Chromosome Mapping Cricetinae DNA Replication Female Ovum/metabolism Xenopus/metabolism
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Gilbert D M
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110-1199, USA.
Miyazawa H
DePamphilis M L
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.15.6.2942
Published
1995-06-00
Pages
2942-54
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC230525
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.15.6.2942 PMC Full Text View journal details
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