Characterization of the RAD10 gene of Saccharomyces cerevisiae and purification of Rad10 protein

Lee Bardwell, Helmut Burtscher, William A. Weiss, Charles M. Nicolet, Errol C. Friedberg

Research output: Contribution to journalArticle

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Abstract

The RAD10 gene of Saccharomyces cerevisiae is one of at least five genes required for damage-specific incision of DNA during nucleotide excision repair. This gene was previously cloned and sequenced [Weiss, W. A., & Friedberg, E. C. (1985) EMBO J. 4, 1575-1582; Reynolds et al. (1985) EMBO J. 4, 3549-3552]. In the present studies, we have mapped one major and three minor transcriptional start sites in the RAD10 gene. The locations of these sites relative to the translational start codon are remarkably similar to those previously identified in the yeast RAD2 gene [Nicolet et al. (1985) Gene 36, 225-234]. The two genes also share common sequences in these regions. However, in contrast to RAD2 [Robinson et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1842-1846], RAD10 is not induced following exposure of cells to the DNA-damaging agent 4-nitroquinoline 1-oxide. Native Rad10 protein and also two different Rad10 fusion proteins are rapidly degraded in most Escherichia coli strains. However, following overexpression of the cloned RAD10 gene in yeast, native Rad10 protein was purified to >90% homogeneity. A catalytic function has not been identified for the purified protein. RAD10 cells (untransformed with the cloned gene) contain fewer than 500 molecules per cell. This is similar to the levels of the UvrA, UvrB, and UvrC nucleotide excision repair proteins in E. coli.

Original languageEnglish (US)
Pages (from-to)3119-3126
Number of pages8
JournalBiochemistry
Volume29
Issue number12
StatePublished - 1990

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Yeast
Purification
Saccharomyces cerevisiae
Genes
Proteins
DNA Repair
Escherichia coli
Repair
Nucleotides
Yeasts
4-Nitroquinoline-1-oxide
Initiator Codon
Escherichia coli Proteins
DNA
Fusion reactions
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bardwell, L., Burtscher, H., Weiss, W. A., Nicolet, C. M., & Friedberg, E. C. (1990). Characterization of the RAD10 gene of Saccharomyces cerevisiae and purification of Rad10 protein. Biochemistry, 29(12), 3119-3126.

Characterization of the RAD10 gene of Saccharomyces cerevisiae and purification of Rad10 protein. / Bardwell, Lee; Burtscher, Helmut; Weiss, William A.; Nicolet, Charles M.; Friedberg, Errol C.

In: Biochemistry, Vol. 29, No. 12, 1990, p. 3119-3126.

Research output: Contribution to journalArticle

Bardwell, L, Burtscher, H, Weiss, WA, Nicolet, CM & Friedberg, EC 1990, 'Characterization of the RAD10 gene of Saccharomyces cerevisiae and purification of Rad10 protein', Biochemistry, vol. 29, no. 12, pp. 3119-3126.
Bardwell, Lee ; Burtscher, Helmut ; Weiss, William A. ; Nicolet, Charles M. ; Friedberg, Errol C. / Characterization of the RAD10 gene of Saccharomyces cerevisiae and purification of Rad10 protein. In: Biochemistry. 1990 ; Vol. 29, No. 12. pp. 3119-3126.
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