View the Current Issue

Article

  • The EMBO Journal (2004) 23, 3886 - 3896
  • doi:10.1038/sj.emboj.7600383

Published online: 9 September 2004

Rad18 guides poleta to replication stalling sites through physical interaction and PCNA monoubiquitination

Kenji Watanabe1,2,a, Satoshi Tateishi1,a, Michio Kawasuji2, Toshiki Tsurimoto3, Hirokazu Inoue4 and Masaru Yamaizumi1

  1. Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan
  2. Department of Cardiovascular Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
  3. Department of Biology, School of Sciences, Kyushu University, Hakozaki, Higashi-ku, Fukuoka, Japan
  4. Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Japan

Correspondence to:

Masaru Yamaizumi, Institute of Molecular Embryology and Genetics, Kumamoto University, Kuhonji 4-24-1, Kumamoto 862-0976, Japan. Tel.: +81 96 373 6601; Fax: +81 96 373 6604; E-mail: yamaizm@gpo.kumamoto-u.ac.jp

aThese authors contributed equally to this work

Received 3 May 2004; Accepted 4 August 2004

The DNA replication machinery stalls at damaged sites on templates, but normally restarts by switching to a specialized DNA polymerase(s) that carries out translesion DNA synthesis (TLS). In human cells, DNA polymerase eta (poleta) accumulates at stalling sites as nuclear foci, and is involved in ultraviolet (UV)-induced TLS. Here we show that poleta does not form nuclear foci in RAD18-/- cells after UV irradiation. Both Rad18 and Rad6 are required for poleta focus formation. In wild-type cells, UV irradiation induces relocalization of Rad18 in the nucleus, thereby stimulating colocalization with proliferating cell nuclear antigen (PCNA), and Rad18/Rad6-dependent PCNA monoubiquitination. Purified Rad18 and Rad6B monoubiquitinate PCNA in vitro. Rad18 associates with poleta constitutively through domains on their C-terminal regions, and this complex accumulates at the foci after UV irradiation. Furthermore, poleta interacts preferentially with monoubiquitinated PCNA, but poldelta does not. These results suggest that Rad18 is crucial for recruitment of poleta to the damaged site through protein–protein interaction and PCNA monoubiquitination.

  • Keywords:

    • PCNA,
    • polymerase eta,
    • Rad6,
    • Rad18,
    • ubiquitination