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Identification of a site required for DNA replication fork blocking activity in the rRNA gene cluster in Saccharomyces cerevisiae

Molecular and General Genetics MGG volume 233, pages 355–362 (1992)Cite this article

Summary

The yeast genome has DNA replication fork blocking sites, that we have named sog sites, in the ribosomal RNA gene (rDNA) cluster. These are located at the 3′ end of the 35S rRNA transcription unit and they block replication fork movement in a direction opposite to that of RNA polymerase I. We cloned this replication blocking site into a YEp-type plasmid and analyzed DNA replication intermediates, using two-dimensional (2D) agarose gel electrophoresis. The blocking activity remained even on a plasmid not involved in 35S rRNA transcription and inhibited fork movement in the same polar fashion as on the yeast chromosome. To define the site further, smaller fragments were subcloned into the YEp-type plasmid. A small 109 by region exhibited sog activity and was located near the enhancer region for 35S rRNA transcription. It overlaps an essential element of the recombinational hot spot HOT1.

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Abbreviations

Ap:

ampicillin

Tc:

tetracycline

URA :

uracil

bp:

base pair(s)

kb:

kilobase pair(s)

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Affiliations

  1. Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 812, Maidashi, Fukuoka, Japan

    Takehiko Kobayashi

  2. Laboratory of Gene Expression and Regulation, National Institute for Basic Biology, 38 Nishigonaka, 444, Myodaijicho, Okazaki, Japan

    Masumi Hidaka & Takashi Horiuchi

  3. Department of Microbiology, School of Medicine, Keio University, 35 Shinanomachi, 106, Shinjukuku, Tokyo, Japan

    Masafumi Nishizawa

Authors
  1. Takehiko Kobayashi
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  2. Masumi Hidaka
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  3. Masafumi Nishizawa
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  4. Takashi Horiuchi
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Communicated by M. Sekiguchi

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Kobayashi, T., Hidaka, M., Nishizawa, M. et al. Identification of a site required for DNA replication fork blocking activity in the rRNA gene cluster in Saccharomyces cerevisiae . Molec. Gen. Genet. 233, 355–362 (1992). https://doi.org/10.1007/BF00265431

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  • DOI: https://doi.org/10.1007/BF00265431

Key words

  • Saccharomyces cerevisiae
  • Replication fork block
  • rRNA gene
  • 2 gm Plasmid
  • 2D agarose gel electrophoresis