Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product. |
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Authors: | B Connolly C A Parsons F E Benson H J Dunderdale G J Sharples R G Lloyd and S C West |
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Institution: | Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, United Kingdom. |
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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. |
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