RecQ4 promotes the conversion of the pre-initiation complex at a site-specific origin for DNA unwinding in xenopus egg extracts

Yosuke Sanuki, Yumiko Kubota, Masato T. Kanemaki, Tatsuro S. Takahashi, Satoru Mimura, Haruhiko Takisawa

研究成果: Contribution to journalArticle査読

9 被引用数 (Scopus)

抄録

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 a 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.

本文言語英語
ページ(範囲)1010-1023
ページ数14
ジャーナルCell Cycle
14
7
DOI
出版ステータス出版済み - 4 1 2015
外部発表はい

All Science Journal Classification (ASJC) codes

  • 分子生物学
  • 発生生物学
  • 細胞生物学

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