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

• Published in: The Journal of cell biology 1992-10, Vol.119 (1), p.1-15
• Main Authors: Adachi, Yasuhisa, Laemmli, Ulrich K.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies; Base Sequence; Biological and medical sciences; Blotting, Western; Cell cycle; Cell Extracts; Cell Nucleus - chemistry; Cell Nucleus - metabolism; Cellular biology; Chromatin; Chromosomes; Cloning, Molecular; Deoxyribonucleic acid; DNA; DNA - biosynthesis; DNA - genetics; DNA Helicases; DNA replication; DNA Replication - physiology; DNA-Binding Proteins - analysis; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; Eggs; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Generally accepted auditing standards; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Nuclear membrane; Oocytes - chemistry; Oocytes - metabolism; Ova; Proteins; Replication; Replication Protein A; Sequence Homology, Nucleic Acid; Spermatozoa; Xenopus laevis - genetics; Xenopus laevis - metabolism
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.119.1.1

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.