The full-length Saccharomyces cerevisiae Sgs1 protein is a vigorous DNA helicase that preferentially unwinds holliday junctions

Petr Cejka, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

74 Citations (Scopus)

Abstract

The highly conserved RecQ family of DNA helicases has multiple roles in the maintenance of genome stability. Sgs1, the single RecQ homologue in Saccharomyces cerevisiae, acts both early and late during homologous recombination. Here we present the expression, purification, and biochemical analysis of full-length Sgs1. Unlike the truncated form of Sgs1 characterized previously, full-length Sgs1 binds diverse single-stranded and double-stranded DNA substrates, including DNA duplexes with 5′- and 3′-single- stranded DNA overhangs. Similarly, Sgs1 unwinds a variety of DNA substrates, including blunt-ended duplex DNA. Significantly, a substrate containing a Holliday junction is unwound most efficiently. DNA unwinding is catalytic, requires ATP, and is stimulated by replication protein A. Unlike RecQ homologues from multicellular organisms, Sgs1 is remarkably active at picomolar concentrations and can efficiently unwind duplex DNA molecules as long as 23,000 base pairs. Our analysis shows that Sgs1 resembles Escherichia coli RecQ protein more than any of the human RecQ homologues with regard to its helicase activity. The full-length recombinant protein will be invaluable for further investigation of Sgs1 biochemistry.

Original languageEnglish (US)
Pages (from-to)8290-8301
Number of pages12
JournalJournal of Biological Chemistry
Volume285
Issue number11
DOIs
StatePublished - Mar 12 2010

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Cruciform DNA
DNA Helicases
Saccharomyces cerevisiae Proteins
Yeast
DNA
RecQ Helicases
Proteins
Substrates
Replication Protein A
Biochemistry
Genomic Instability
Escherichia coli Proteins
Single-Stranded DNA
Homologous Recombination
Recombinant Proteins
Base Pairing
Escherichia coli
Purification
Saccharomyces cerevisiae
Genes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

The full-length Saccharomyces cerevisiae Sgs1 protein is a vigorous DNA helicase that preferentially unwinds holliday junctions. / Cejka, Petr; Kowalczykowski, Stephen C.

In: Journal of Biological Chemistry, Vol. 285, No. 11, 12.03.2010, p. 8290-8301.

Research output: Contribution to journalArticle

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