Purification of Rad1 protein from Saccharomyces cerevisiae and further characterization of the Rad1/Rad10 endonuclease complex

Alan E. Tomkinson, A. Jane Bardwell, Nancy Tappe, William Ramos, Errol C. Friedberg

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

The yeast recombination and repair proteins Rad1 and Rad10 associate with a 1:1 stoichiometry to form a stable complex with a relative molecular mass of 190 kDa. This complex, which has previously been shown to degrade single-stranded DNA endonucleolytically also cleaves supercoiled duplex DNA molecules. In this reaction, supercoiled (form I) molecules are rapidly converted to nicked, relaxed (form II) molecules, presumably as a result of nicking at transient single-stranded regions in the supercoiled DNA. A high enzyme concentrations, there is a slow conversion of the form II molecules to linear (form III) molecules. The Rad1/Rad10 endonuclease does not preferentially cleave UV-irradiated DNA and has no detectable exonuclease activity. The nuclease activity of the Rad1/Rad10 complex is consistent with the predicted roles of the RAD1 and RAD10 genes of Saccharomyces cerevisiae in both the incision events of nucleotide excision repair and the removal of nonhomologous 3′ single strands during intrachromosomal recombination between repeated sequences. In these pathways, the specificity and reactivity of the Rad1/ Rad10 endonuclease will probably be modulated by further protein-protein interactions.

Original languageEnglish (US)
Pages (from-to)5305-5311
Number of pages7
JournalBiochemistry
Volume33
Issue number17
StatePublished - 1994

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Saccharomyces cerevisiae Proteins
Endonucleases
Purification
Superhelical DNA
Molecules
Recombinational DNA Repair
Exonucleases
Proteins
Single-Stranded DNA
Yeast
Repair
DNA Repair
Genetic Recombination
Saccharomyces cerevisiae
Yeasts
DNA
Molecular mass
Stoichiometry
Enzymes
Nucleotides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification of Rad1 protein from Saccharomyces cerevisiae and further characterization of the Rad1/Rad10 endonuclease complex. / Tomkinson, Alan E.; Bardwell, A. Jane; Tappe, Nancy; Ramos, William; Friedberg, Errol C.

In: Biochemistry, Vol. 33, No. 17, 1994, p. 5305-5311.

Research output: Contribution to journalArticle

Tomkinson, Alan E. ; Bardwell, A. Jane ; Tappe, Nancy ; Ramos, William ; Friedberg, Errol C. / Purification of Rad1 protein from Saccharomyces cerevisiae and further characterization of the Rad1/Rad10 endonuclease complex. In: Biochemistry. 1994 ; Vol. 33, No. 17. pp. 5305-5311.
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