Rad3 protein of Saccharomyces cerevisiae: Overexpression and preliminary characterization using specific antibodies

Louie Naumovski, Errol C. Friedberg

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

The cloned RAD3 gene of Saccharomyces cerevisiae was tailored into expression vectors for overexpression of Rad3 protein in Escherichia coli and in yeast. In both organisms the overexpressed protein is detected as a species of molecular weight ca. 90 kDa, the size expected from the sequence of the cloned gene. The protein overexpressed in E. coli is largely insoluble; however the insoluble fraction was used to generate affinity-purified polyclonal antisera which proved to be powerful reagents for the initial characterization of Rad3 protein expressed in yeast. These studies showed that: (1) when overexpressed in yeast most of the Rad3 protein is detected in the soluble fraction of cell extracts; (2) endogenous Rad3 protein is untransformed cells is also ca. 90 kDa in size and is located in the cell nucleus; (3) Rad3/β-galactosidase fusion protein partially purified on an affinity matrix is associated with DNA-dependent ATPase activity that is inhibited in the presence of anti-Rad3 antibodies, suggesting that Rad3 protein is an ATPase; and (4) Rad3 antibodies cross-react with two electrophoretically distinguishable polypeptides present in the nuclear fraction of human cells, and with a single polypeptide in extracts of Drosophila cell.

Original languageEnglish (US)
Pages (from-to)400-408
Number of pages9
JournalMGG Molecular & General Genetics
Volume213
Issue number2-3
DOIs
StatePublished - Aug 1988

Keywords

  • DNA repair
  • Fusion proteins
  • RAD3 gene expression
  • Yeast

ASJC Scopus subject areas

  • Genetics

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