Saccharomyces cerevisiae proteins involved in hybrid DNA formation in vitro

W. D. Heyer, A. W. Johnson, D. N. Norris, D. Tishkoff, R. D. Kolodner

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

RecA-like activities that can form hybrid DNA in vitro have been identified in a wide variety of organisms. We have previously described the strand exchange protein 1 (SEP1) from the yeast Saccharomyces cerevisiae that can form hybrid DNA in vitro. Purified as an Mr 132 000 polypeptide, recent molecular and immunological studies have now shown that the native form is an Mr 175 000 polypeptide containing strand exchange activity. The gene encoding SEP1 has been cloned and sequenced. The primary sequence failed to reveal any significant sequence homology to other sequences in data base searches. In vivo SEP1 was found to be essential for normal meiosis as cells containing a homozygous insertion mutation in the SEP1 gene failed to sporulate. In order to identify additional factors that are involved in hybrid DNA formation in S cerevisiae, we used an in vitro stimulation assay to identify proteins that reconstitute strand exchange activity in reactions containing limiting amounts of SEP1. We have identified two proteins that functionally interact with SEP1. First, an Mr 34 000 single-stranded DNA binding protein stimulated the reaction by lowering the requirement for SEP1 about 3-4 fold. This protein is a fragment of the large subunit of a hetero-trimeric complex called yRP-A (yRF-A) which is thought to be the functional eukaryotic equivalent of single-stranded DNA binding proteins in prokaryotes. The gene encoding this protein (RPA1) is essential for growth. Second, an Mr 33 000 polypeptide, termed Stimulatory Factor 1 (SF1), dramatically stimulated the SEP1 catalyzed reaction by lowering the requirement for SEP1 about 300 fold. This protein specifically stimulated SEP1 in a way that is quantitatively and qualitatively different from previously found stimulation of strand exchange proteins.

Original languageEnglish (US)
Pages (from-to)269-276
Number of pages8
JournalBiochimie
Volume73
Issue number2-3
DOIs
StatePublished - 1991
Externally publishedYes

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Keywords

  • hybrid DNA
  • recombination
  • S cerevisiae
  • strand exchange

ASJC Scopus subject areas

  • Biochemistry

Cite this

Heyer, W. D., Johnson, A. W., Norris, D. N., Tishkoff, D., & Kolodner, R. D. (1991). Saccharomyces cerevisiae proteins involved in hybrid DNA formation in vitro. Biochimie, 73(2-3), 269-276. https://doi.org/10.1016/0300-9084(91)90212-J