Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product

Bernadette Connolly, C A Parsons, F E Benson, H J Dunderdale, G J Sharples, R G Lloyd, S C West

Research output: Contribution to journalArticle

142 Citations (Scopus)

Abstract

In previous studies, Holliday junctions generated during RecA-mediated strand-exchange reactions were resolved by fractionated Escherichia coli extracts. We now report the specific binding and cleavage of synthetic Holliday junctions (50 base pairs long) by a fraction purified by chromatography on DEAE-cellulose, phosphocellulose, and single-stranded DNA-cellulose. The cleavage reaction provided a sensitive assay with which to screen extracts prepared from recombination/repair-deficient mutants. Cells with mutations in ruvC lack the nuclease activity that cleaves synthetic Holliday junctions in vitro. This deficiency was restored by a multicopy plasmid carrying a ruvC+ gene that overexpressed junction-resolving activity. The UV sensitivity and deficiency in recombinational repair of DNA exhibited by ruv mutants lead us to suggest that RuvC resolves Holliday junctions in vivo.
Original languageEnglish
Pages (from-to)6063-6067
Number of pages5
JournalPNAS
Volume88
Issue number14
Publication statusPublished - 15 Jul 1991

Keywords

  • Bacterial Proteins
  • Bacteriophage phi X 174
  • Base Sequence
  • DNA Replication
  • DNA, Bacterial
  • DNA, Viral
  • Endodeoxyribonucleases
  • Escherichia coli
  • Escherichia coli Proteins
  • Gene Expression
  • Genes, Bacterial
  • Molecular Sequence Data
  • Oligonucleotide Probes
  • Plasmids

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