FP6 priority

1.1.1   Genomics and Biotechnology for Health

1.1.1.1

Title of the proposal

Structure-function analysis of the DNA repair proteins

Institute

Slovak Academy of Sciences, Cancer Research Institute
Vlarska 7, 83391 Bratislava 37, Slovak Republic

Contact

Research subject for a potential FP6 project

The Xpb/Ercc3 helicase is essential for both nucleotide excision repair (NER) and transcription as a subunit of a multifunctional RNA polymerase II general initiation factor TFIIH. In NER it unwinds DNA around the damage by about 25-30 base pairs forming the open complex. This allows two structure-specific endonucleases, Xpg and Ercc1-Xpf, to perform dual incision. In transcription Xpb functions at multiple steps to promote efficient initiation and promoter escape by RNA polymerase II. Mutations in the Xpb gene in humans give rise to two distinct, autosomal recessive disorders, with a striking clinical heterogeneity: xeroderma pigmentosum associated with Cockayne’s syndrome (XP/CS) and trichothiodystrophy (TTD). Out of five known patients, three XP-B patients from two families have combined symptom of XP/CS and two other siblings have TTD. Their cells exhibit different levels of DNA repair and transcription. The three respective mutations of Xpb gene are known. Since Xpb defective human disease is extremely rare, Chinese hamster (CHO) mutant cell lines belonging to the third rodent complementation group (CG3) are a unique resource for analyzing structure-function relationships in the Xpb/Ercc3 protein. There are nine CHO mutant cell lines assigned to CG3 that have never been characterized. To analyze domains of the Xpb protein, we propose to: (1) determine the nucleotide sequence and identify the mutations in the Xpb gene, (2) compare the newly identified Xpb mutations with the known mutations in XP-B patients and with their effects on the level of NER, (3) correlate the sites of the particular mutations with the DNA repair phenotypes. Studying these historically unique hamster mutant cell lines from CG3 is a valuable, efficient approach for understanding the properties of the human Xpb/Ercc3 protein, and data obtained from these studies is expected to reveal important new insights into the functional domain(s) within Xpb/Ercc3 protein.

Recent international cooperation of the research team

Leiden University, Leiden, The Netherlands; CNRS-CEA, Paris, France; Rowett Research Institute, Aberdeen, United Kingdom; Paterson Institute for Cancer Research, Manchester,United Kingdom; Lawrence Livermore National Laboratory, Livermore, USA; National Institutes onAging, Baltimore, USA

Proposer´s relevant publications related to the research subject

1. Bartosova Z., Horak I.D., Pirsel M., Bohr V.A.: Lack of correlation between repair of DNA interstrand cross-links and differential sensitivity of G0 and proliferating CD4+ lymphocytes towards cisplatin. Neoplasma 1999, 46: 342-348.
2. Bartosova Z, Pirsel M, Reinhold W, Stetler-Stevenson M, Zajac-Kaye M, May A, Horak ID, Bohr VA.: Gene-specific repair in human CD4+ lymphocytes reflects transcription and proliferation. Mutat Res. 1996, 363: 191-199.
3. Pirsel M, Bohr VA.: Methyl methanesulfonate adduct formation and repair in the DHFR gene and in mitochondrial DNA in hamster cells. Carcinogenesis 1993, 14: 2105-2108.
4. Stevnsner T, May A, Petersen LN, Larminat F, Pirsel M, Bohr VA.: Repair of ribosomal RNA genes in hamster cells after UV irradiation, or treatment with cisplatin or alkylating agents. Carcinogenesis 1993, 14: 1591-1596.