Identification of nuclear pre-replication centers poised for DNA synthesis in Xenopus egg extracts: immunolocalization study of replication protein A.
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.
Adachi
Y
Y
Department of Biochemistry, University of Geneva, Switzerland.
Laemmli
U K
UK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002457:Cell Extracts; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004265:DNA Helicases; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D009865:Oocytes / Q000737:chemistry / Q000378:metabolism; D051716:Replication Protein A; D012689:Sequence Homology, Nucleic Acid; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D051716:Replication Protein A; C076713:P70 protein, Xenopus; D004247:DNA; D004265:DNA Helicases