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  • The EMBO Journal (1999) 18, 3491 - 3501
  • doi:10.1093/emboj/18.12.3491

Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity

Chikahide Masutani1, Marito Araki1,2,3, Ayumi Yamada1,2, Rika Kusumoto1,4, Tomokazu Nogimori1,2, Takafumi Maekawa1, Shigenori Iwai5 and Fumio Hanaoka1,3

  1. Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan
  2. The Graduate School of Pharmaceutical Science, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
  3. The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama, 351-0198, Japan
  4. Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara, 630-01, Japan
  5. Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka, 565-0874, Japan

Correspondence to:

Fumio Hanaoka, E-mail: fhanaoka@imcb.osaka-u.ac.jp

Received 6 April 1999; Accepted 29 April 1999; Revised 29 April 1999

Xeroderma pigmentosum variant (XP-V) represents one of the most common forms of this cancer-prone DNA repair syndrome. Unlike classical XP cells, XP-V cells are normal in nucleotide excision repair but defective in post-replication repair. The precise molecular defect in XP-V is currently unknown, but it appears to be a protein involved in translesion synthesis. Here we established a sensitive assay system using an SV40 origin-based plasmid to detect XP-V complementation activity. Using this system, we isolated a protein from HeLa cells capable of complementing the defects in XP-V cell extracts. The protein displays novel DNA polymerase activity which replicates cyclobutane pyrimidine dimer-containing DNA templates. The XPV polymerase activity was dependent on MgCl2, sensitive to NEM, moderately sensitive to KCl, resistant to both aphidicolin and ddTTP, and not stimulated by PCNA. In glycerol density gradients, the activity co-sedimented with a 54 kDa polypeptide at 3.5S, indicating that the monomeric form of this polypeptide was responsible for the activity. The protein factor corrected the translesion defects of extracts from three XPV cell strains. Bypass DNA synthesis by the XP-V polymerase occurred only in the presence of dATP, indicating that it can incorporate only dATP to bypass a di-thymine lesion.

  • Keywords:

    • cyclobutane pyrimidine dimer,
    • DNA polymerase,
    • DNA replication,
    • error-free translesion synthesis,
    • xeroderma pigmentosum variant