Purification and characterization of the Saccharomyces cerevisiae RAD1/RAD10 endonuclease

Patrick Sung, Paul Reynolds, Louise Prakash, Satya Prakash

Research output: Contribution to journalArticle

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Abstract

The Saccharomyces cerevisiae RAD1 and RAD10 genes are required for the incision step of excision repair, and in addition, they function in mitotic recombination. The RAD1 and RAD10 proteins are associated in a tight complex, and genetic studies have indicated that complex formation is essential for the RAD1/ RAD10 controlled biological activities. We had previously purified the RAD10 protein to near homogeneity from yeast and shown that it is a DNA-binding protein with a strong preference for single-stranded DNA. In this study, we purify the RAD1 protein to near homogeneity from yeast and show that it also binds single-stranded DNA preferentially and that the RAD1/ RAD10 complex possesses an endonuclease activity. We characterize the RAD1/RAD10 endonuclease activity on both single-stranded and double-stranded DNAs, using agarose gel electrophoresis and trichloroacetic acid precipitation. The RAD1/RAD10 nuclease exhibits a much higher level of activity on single-stranded DNA than double-stranded DNA. The susceptibility of double-stranded DNA to nicking by the RAD1/RAD10 enzyme is markedly dependent on the degree of negative superhelicity, such that a 15-fold increase in nicking rate is observed from superhelical state σ = zero to σ = -0.08. The enzyme produces 3′-hydroxyl and 5′-phosphate termini on both single-and double-stranded DNAs. We discuss the role of RAD1/RAD10 endonuclease in nucleotide excision repair and in mitotic recombination.

Original languageEnglish (US)
Pages (from-to)26391-26399
Number of pages9
JournalJournal of Biological Chemistry
Volume268
Issue number35
StatePublished - Dec 15 1993

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Endonucleases
Single-Stranded DNA
Yeast
Purification
Saccharomyces cerevisiae
DNA
DNA Repair
Genetic Recombination
Repair
Yeasts
Trichloroacetic Acid
Proteins
Agar Gel Electrophoresis
DNA-Binding Proteins
Enzymes
Bioactivity
Electrophoresis
Hydroxyl Radical
Sepharose
Nucleotides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and characterization of the Saccharomyces cerevisiae RAD1/RAD10 endonuclease. / Sung, Patrick; Reynolds, Paul; Prakash, Louise; Prakash, Satya.

In: Journal of Biological Chemistry, Vol. 268, No. 35, 15.12.1993, p. 26391-26399.

Research output: Contribution to journalArticle

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