title: RecQ4 promotes the conversion of the pre-initiation complex at a site-specific origin for DNA unwinding in Xenopus egg extracts.
abstract: Eukaryotic DNA replication is initiated through stepwise assembly of evolutionarily conserved replication proteins onto replication origins, but how the origin DNA is unwound during the assembly process remains elusive. Here, we established a site-specific origin on a plasmid DNA, using in vitro replication systems derived from Xenopus egg extracts. We found that the pre-replicative complex (pre-RC) was preferentially assembled in the vicinity of GAL4 DNA-binding sites of the plasmid, depending on the binding of Cdc6 fused with a GAL4 DNA-binding domain in Cdc6-depleted extracts. Subsequent addition of nucleoplasmic S-phase extracts to the GAL4-dependent pre-RC promoted initiation of DNA replication from the origin, and components of the pre-initiation complex (pre-IC) and the replisome were recruited to the origin concomitant with origin unwinding. In this replication system, RecQ4 is dispensable for both recruitment of Cdc45 onto the origin and stable binding of Cdc45 and GINS to the pre-RC assembled plasmid. However, both origin binding of DNA polymerase α and unwinding of DNA were diminished upon depletion of RecQ4 from the extracts. These results suggest that RecQ4 plays an important role in the conversion of pre-ICs into active replisomes requiring the unwinding of origin DNA in vertebrates.
authors.affiliation: a Department of Biological Sciences; Graduate School of Science ; Osaka University ; Toyonaka , Osaka , Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Sanuki
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Kubota
authors.forename: Masato T
authors.initials: MT
authors.lastname: Kanemaki
authors.forename: Tatsuro S
authors.initials: TS
authors.lastname: Takahashi
authors.forename: Satoru
authors.initials: S
authors.lastname: Mimura
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Takisawa
mesh_terms: D000818:Animals; D001665:Binding Sites; D002457:Cell Extracts; D002474:Cell-Free System; D002478:Cells, Cultured; D004261:DNA Replication; D009865:Oocytes; D010957:Plasmids / Q000235:genetics; D053484:RecQ Helicases / Q000502:physiology*; D018741:Replication Origin; D014157:Transcription Factors / Q000502:physiology; D029867:Xenopus Proteins / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D002457:Cell Extracts; D014157:Transcription Factors; D029867:Xenopus Proteins; D053484:RecQ Helicases
keywords: DNA unwinding; RecQ4; Xenopus egg extracts; eukaryotic DNA replication; pre-IC; pre-RC; site-specific origin