George, Helen Marion; (2000) Characterisation of the RuvB branch migration motor. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Escherichia coli Ruv proteins process Holliday junction intermediates during the recombinational repair of DNA damage. RuvA and RuvB promote branch migration, a reaction responsible for the formation and extension of heteroduplex DNA, whilst RuvC mediates junction resolution to form recombinant products. A tripartite RuvAB-Holliday junction complex has been observed by electron microscopy. RuvA is bound to the crossover and is flanked by two RuvB hexameric rings threaded on opposite arms of the junction. The RuvB rings provide a novel dual motor for branch migration, using the energy from ATP hydrolysis to pump duplex DNA out through their central cavities. RuvB belongs to a family of hexameric helicases and, in the presence of RuvA, exhibits a 5' to 3' DNA helicase activity in vitro. To investigate the role of DNA unwinding in branch migration, site-directed mutagenesis has been used to knock-out a residue in RuvB that is important for its helicase activity. The resultant protein, RuvBD113E, has been purified and characterised in vivo and in vitro. Despite being severely compromised in its DNA helicase activity, RuvBD113E was able to hydrolyse ATP and promote branch migration in the presence of RuvA. The results indicate that extensive helicase activity is not required for RuvAB-mediated branch migration. It is possible that transient opening of the duplex arms within the RuvB rings may occur which would provide a single strand for unidirectional translocation. However, introduction of an interstrand cross-link into a synthetic junction did not affect the efficiency of RuvAB-mediated branch migration. Based on the results of these studies, and on observations made with the other hexameric helicases, models for DNA translocation by RuvB are proposed.

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