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PMID: 8947545 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts.

The Journal of cell biology ·Vol. 135 ·No. 5 ·1996-12-00 ·Pages 1207-18

Lawlis SJ, Keezer SM, Wu JR, Gilbert DM

Abstract

Xenopus egg extracts initiate DNA replication specifically at the dihydrofolate reductase (DHFR) origin locus with intact nuclei from late G1-phase CHO cells as a substrate, but at nonspecific sites when purified DNA is assembled by the extract into an embryonic nuclear structure. Here we show that late G1-phase CHO nuclei can be cycled through an in vitro Xenopus egg mitosis, resulting in the assembly of an embryonic nuclear envelope around G1-phase chromatin. Surprisingly, replication within these chimeric nuclei initiated at a novel specific site in the 5' region of the DHFR structural gene that does not function as an origin in cultured CHO cells. Preferential initiation at this unusual site required topoisomerase II-mediated chromosome condensation during mitosis. Nuclear envelope breakdown and reassembly in the absence of chromosome condensation resulted in nonspecific initiation. Introduction of condensed chromosomes from metaphase-arrested CHO cells directly into Xenopus egg extracts was sufficient to elicit assembly of chimeric nuclei and preferential initiation at this same site. These results demonstrate clearly that chromosome architecture can determine the sites of initiation of replication in Xenopus egg extracts, supporting the hypothesis that patterns of initiation in vertebrate cells are established by higher order features of chromosome structure.

MeSH Terms
Animals CHO Cells Cell Extracts Cell Nucleus/metabolism,ultrastructure Chromosomes/metabolism,ultrastructure Cricetinae DNA Replication G1 Phase Genes Metaphase Mitosis Nuclear Envelope/metabolism Ovum Replication Origin Tetrahydrofolate Dehydrogenase/genetics Xenopus
Chemicals
Cell Extracts Tetrahydrofolate Dehydrogenase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Lawlis S J
Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse 13210, USA.
Keezer S M
Wu J R
Gilbert D M
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.135.5.1207
Published
1996-12-00
Pages
1207-18
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2121087
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.135.5.1207 PMC Full Text View journal details
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