SummaryNucleoprotein complexes present challenges to genome stability by acting as potent blocks to replication. One attractive model of how such conflicts are resolved is direct targeting of blocked forks by helicases with the ability to displace the blocking protein-DNA complex. We show that Rep and UvrD each promote movement of E. coli replisomes blocked by nucleoprotein complexes in vitro, that such an activity is required to clear protein blocks (primarily transcription complexes) in vivo, and that a polarity of translocation opposite that of the replicative helicase is critical for this activity. However, these two helicases are not equivalent. Rep but not UvrD interacts physically and functionally with the replicative helicase. In contrast, UvrD likely provides a general means of protein-DNA complex turnover during replication, repair, and recombination. Rep and UvrD therefore provide two contrasting solutions as to how organisms may promote replication of protein-bound DNA.

The Escherichia coli DNA binding protein RuvA acts in concert with the helicase RuvB to drive branch migration of Holliday intermediates during recombination and DNA repair. The atomic structure of RuvA was determined at a resolution of 1.9 angstroms. Four monomers of RuvA are related by fourfold symmetry in a manner reminiscent of a four-petaled flower. The four DNA duplex arms of a Holliday junction can be modeled in a square planar configuration and docked into grooves on the concave surface of the protein around a central pin that may facilitate strand separation during the migration reaction. The model presented reveals how a RuvAB-junction complex may also accommodate the resolvase RuvC.

The ruvA, ruvB, and ruvC genes of Escherichia coli provide activities that catalyze branch migration and resolution of Holliday junction intermediates in recombination. Mutation of any one of these genes interferes with recombination and reduces the ability of the cell to repair damage to DNA. A suppressor of ruv mutations was identified on the basis of its ability to restore resistance to mitomycin and UV light and to allow normal levels of recombination in a recBC sbcBC strain carrying a Tn10 insertion in ruvA. The mutation responsible was located at 12.5 min on the genetic map and defines a new locus which has been designated rus. The rus suppressor works just as well in recBC sbcA and rec+ sbc+ backgrounds and is not allele specific. Mutations in ruvB and ruvC are suppressed to an intermediate level, except when ruvA is also inactive, in which case suppression is complete. In all cases, suppression depends on RecG protein, a DNA-dependent ATPase that catalyzes branch migration of Holliday junctions. The rus mutation activates an additional factor that probably works with RecG to process Holliday junction intermediates independently of the RuvAB and RuvC proteins. The possibility that this additional factor is a junction-specific resolvase is discussed.