XERODERMA PIGMENTOSUM VARIANT: GENE AND PROTEIN
Institute for Molecular and Cellular Biology, Osaka University, and CREST, Japan Science and Technology Corporation
1-3 Yamada-oka, Suita, Osaka 565-0871, and RIKEN, 2-1 Hirosawa, Wako, Saitama 351-01 98, Japan
(Tel: 81-6-6879-7975 Fax: 81-6-6877-9382 E-mail: firstname.lastname@example.org)
Xeroderma pigmentosum (XP) is an autosomal recessive human disease characterized by sunlight sensitivity, cutaneous and ocular deterioration, and premature malignant skin neoplasms after exposure to sunlight. XP has been classified into eight complementation groups, XP-A to XP-G and XP-V. Cells from XP-A to XP-G patients have defects in the process of nucleotide excision repair (NER), which eliminates a wide variety of structurally unrelated lesions, including ultraviolet light (UV)-induced cyclobutane pyrimidine dimers (CPD) and (6-4)photoproducts, as well as certain chemical adducts. The genes and proteins of XP groups A, B, C, D, F, and G have been isolated and found to represent some of the subunits of the core NER machinery. In contrast, cells belonging to the eighth group, XP variant (XP-V), are NER-proficient but display abnormal DNA replication, including reduced ability to elongate nascent DNA strands on UV irradiated DNA. Thus, the XPV gene product is likely to be involved in the process of DNA replication on damaged DNA known as post-replication repair, but not in NER. In fact, using a plasmid DNA containing a single CPD on the leading strand, some groups reported that extracts from XP-V cells lacked lesion bypass. However, isolation of the protein which complements the defect of bypass replication of the extracts from XP-V cells was difficult mainly because of the unclear nature of the assay.
We constructed a sensitive assay system to detect an activity that corrects defects of extracts from XP-V cells, purified the XP-V correcting protein from HeLa cells, and found that the protein has a novel DNA polymerase activity. Furthermore, we found by molecular cloning that this polymerase is a human homolog of the yeast RAD30 protein, DNA polymerase!!!.This provides for the first time a molecular explanation for the unique complementation group of the famous cancer-prone genetic disease, xeroderma pigmentosum variant.
Human pol!!!can catalyze translesion synthesis past not only CPD but also an abasic site analog, N-2-acetylaminofluorene-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. Using steady-state kinetics, we determined the accuracy of DNA synthesis by human pol!!!, and found that the accuracy of DNA synthesis opposite the damaged DNA by this enzyme is nearly equal with that opposite non-damaged DNA. Mechanisms of apparent accurate translesion synthesis by the human pol!!!will be discussed.
(1) C. Masutani, M. Araki, A. Yamada, R. Kusumoto, T. Nogimori, T. Maekawa, S. Iwai, and F. Hanaoka (1999) Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa ceils has a thymine dimer bypass DNA polymerase activity. EMBO J.
(2) C. Masutani, R. Kusumoto, A. Yamada, N. Dohmae, M. Yokoi, M. Yuasa, M. Araki, S. Iwai, K. Takio, and F. Hanaoka (1999) The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase!!!. Nature 399:700-704
(3) T. Matsuda, K. Bebenek, C. Masutani, F. Hanaoka, and T. A. Kunkel (2000) Low fidelity DNA synthesis by human DNA polymerase!!!. Nature 404:1011-1013
(4) C. Masutani, R. Kusumoto, S. Iwai, and F. Hanaoka (2000) Mechanisms of accurate translesion synthesis by human DNA polymerase!!!. EMBO J. 19:3100-3109
Fumio Hanaoka PhD
1968 BSc University of Tokyo, Japan
1973 PhD University of Tokyo, Japan
1973-1980 Assistant Professor, University of Tokyo, Japan
1980-1989 Associate Professor, University of Tokyo, Japan
1989-1994 Chief Scientist, RIKEN, Japan
1995- Professor, Osaka University, Japan
Speciality and Special Interest:
Molecular mechanisms of translesion synthesis and nucleotide excision repair, understanding the cellular responses to DNA damages, interactions between cell cycle control and DNA repair.