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EMBO J 1994 Sep 01;1317:4153-64. doi: 10.1002/j.1460-2075.1994.tb06733.x.
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Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts.

Adachi Y , Laemmli UK .

RPA is a cellular, three-subunit, single-stranded (ss) DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. By immunodepletion and complementation, we have identified RPA as an essential factor for cellular DNA replication in Xenopus extracts. RPA assembles post-mitotically on the decondensing chromosomes into numerous subnuclear pre-replication centres (preRCs) which serve, upon formation of the nuclear membrane, as RCs for the initiation of DNA synthesis. By a variety of experiments including the use of isolated components, we demonstrate that an inactive cdc2-cyclin B kinase complex is essential to allow post-mitotic assembly of the preRCs. In contrast, the active cdk2-cyclin A kinase does not impede or facilitate the assembly of preRCs. Digestion analysis using the single-strand-specific P1 nuclease as well as competition experiments with ssDNA, reveal that replication-associated unwinding of the DNA, assisted by RPA, requires the formation of the nuclear membrane. The p21 cdk-interacting protein Cip1 appears to inhibit DNA replication prior to the unwinding DNA step, but after assembly of preRC and nuclear reconstruction.

PubMed ID: 8076611
PMC ID: PMC395338
Article link: EMBO J

Species referenced: Xenopus laevis
Genes referenced: cdk1 cdk2 cdkn1a rpa1

References [+] :
Adachi, Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A. 1992, Pubmed, Xenbase