Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product.
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.
Connolly
B
B
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, United Kingdom.
Parsons
C A
CA
Benson
F E
FE
Dunderdale
H J
HJ
Sharples
G J
GJ
Lloyd
R G
RG
West
S C
SC
D001426:Bacterial Proteins / Q000235:genetics*; D010584:Bacteriophage phi X 174 / Q000235:genetics; D001483:Base Sequence; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004279:DNA, Viral; D029968:Escherichia coli Proteins; D015345:Oligonucleotide Probes; C069806:ruvC protein, E coli; D004706:Endodeoxyribonucleases