RuvAB-mediated branch migration does not involve extensive DNA opening within the RuvB hexamer |
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Authors: | George H Kuraoka I Nauman D A Kobertz W R Wood R D West S C |
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Institution: | Clare Hall Laboratories, Imperial Cancer Research Fund, South Mimms, EN6 3LD, UK. |
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Abstract: | The Escherichia coli RuvA and RuvB proteins promote the branch migration of Holliday junctions during the late stages of homologous recombination and DNA repair (reviewed in 1]). Biochemical and structural studies of the RuvAB-Holliday junction complex have shown that RuvA binds directly to the Holliday junction 2] 3] 4] 5] 6] and acts as a specificity factor that promotes the targeting of RuvB 7] 8], a hexameric ring protein that drives branch migration 9] 10] 11]. Electron microscopic visualisation of the RuvAB complex revealed that RuvA is flanked by two RuvB hexamers, which bind DNA arms that lie diametrically opposed across the junction 8]. ATP-dependent branch migration occurs as duplex DNA is pumped out through the centre of each ring. Because RuvB possesses well-conserved helicase motifs and RuvAB exhibits a 5'-3' DNA helicase activity in vitro 12], the mechanism of branch migration is thought to involve DNA opening within the RuvB ring, which provides a single strand for the unidirectional translocation of the protein along DNA. We have investigated whether the RuvB ring can translocate along duplex DNA containing a site-directed interstrand psoralen crosslink. Surprisingly, we found that the crosslink failed to inhibit branch migration. We interpret these data as evidence against a base-by-base tracking model and suggest that extensive DNA opening within the RuvB ring is not required for DNA translocation by RuvB. |
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