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Identification of Nuclear Pre-replication Centers Poised for DNA Synthesis in Xenopus Egg Extracts: Immunolocalization Study of Replication Protein A

Published inThe Journal of Cell Biology, vol. 119, no. 1, p. 1-15
Publication date1992
Abstract

We demonstrate by immunofluorescence that a 70-kD protein (P70) purified from Xenopus egg extracts is associated with subnuclear foci (about 200) which we propose to be an assembly of DNA pre-replication centers (preRCs). A cDNA encoding this protein reveals that P70 is the Xenopus homologue of replication protein A (RPA also called RF-A). RPA is know to be a cellular, three-subunit single-stranded DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. The punctated preRCs exist transiently; they form post-mitotically during the period of nuclear membrane breakdown and disappear during ongoing DNA replication. P70 is homogeneously associated with chromatin at the later stages of the S-phase and is displaced from chromatin post replication, so that P70 cannot be detected on mitotic chromosomes. Double-immunofluorescence studies using biotin-dUTP demonstrate that initiation of DNA synthesis is confined to preRCs, resulting in the punctated replication pattern observed previously by others. PreRCs form efficiently on decondensed chromatin in membrane-free egg extracts if ATP and divalent cations are present. Our results suggest that preRCs are composed of an assembly of a large number of pre-initiation replication complexes poised for initiation at discreet subnuclear regions prior to nuclear reconstruction and initiation of DNA synthesis.

Funding
  • Swiss National Science Foundation - 31.28667.90
Citation (ISO format)
ADACHI, Yasuhisa, LAEMMLI, Ulrich Karl. Identification of Nuclear Pre-replication Centers Poised for DNA Synthesis in <i>Xenopus</i> Egg Extracts: Immunolocalization Study of Replication Protein A. In: The Journal of Cell Biology, 1992, vol. 119, n° 1, p. 1–15. doi: 10.1083/jcb.119.1.1
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Article (Published version)
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ISSN of the journal0021-9525
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