<b>The directionality of RuvAB‐mediated branch migration: <i>in vitro</i> studies with three‐armed junctions</b>

Hiom, Kevin; Tsaneva, Irina R.; West, Stephen C.

doi:10.1046/j.1365-2443.1996.d01-253.x

Cited by 19 publications

(15 citation statements)

References 26 publications

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“…In contrast, DNA helicase activity and branch migration of three-way junctions appeared unaffected. Branch migration of Y-junctions is driven by a single RuvB hexamer (39). The defined polarity of the DNA helicase activity (50) also implies the involvement of a single RuvB hexamer in this reaction.…”

Section: Discussionmentioning

confidence: 99%

The Role of RuvA Octamerization for RuvAB Function in Vitro and in Vivo

Privezentzev

Keeley

Sigala

et al. 2005

Journal of Biological Chemistry

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RuvA plays an essential role in branch migration of the Holliday junction by RuvAB as part of the RuvABC pathway for processing Holliday junctions in Escherichia coli. Two types of RuvA-Holliday junction complexes have been characterized: 1) complex I containing a single RuvA tetramer and 2) complex II in which the junction is sandwiched between two RuvA tetramers. The functional differences between the two forms are still not clear. To investigate the role of RuvA octamerization, we introduced three amino acid substitutions designed to disrupt the E. coli RuvA tetramer-tetramer interface as identified by structural studies. The mutant RuvA was tetrameric and interacted with both RuvB and junction DNA but, as predicted, formed complex I only at protein concentrations up to 500 nM. We present biochemical and surface plasmon resonance evidence for functional and physical interactions of the mutant RuvA with RuvB and RuvC on synthetic junctions. The mutant RuvA with RuvB showed DNA helicase activity and could support branch migration of synthetic four-way and three-way junctions. However, junction binding and the efficiency of branch migration of four-way junctions were affected. The activity of the RuvA mutant was consistent with a RuvAB complex driven by one RuvB hexamer only and lead us to propose that one RuvA tetramer can only support the activity of one RuvB hexamer. Significantly, the mutant failed to complement the UV sensitivity of E. coli ⌬ruvA cells. These results indicate strongly that RuvA octamerization is essential for the full biological activity of RuvABC.

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Section: Discussionmentioning

confidence: 99%

The Role of RuvA Octamerization for RuvAB Function in Vitro and in Vivo

Privezentzev

Keeley

Sigala

et al. 2005

Journal of Biological Chemistry

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“…A tetramer of RuvA binds Holliday junction DNA and loads two hexameric RuvB helicase rings on opposite arms of the junction (13). The RuvB hexamers pull opposing duplex arms of the junction through the rings with the result being active movement of the branch point along the DNA as it spools across the face of the RuvA tetramer (14,15). The third component of this complex, a dimer of RuvC, binds to the branch point on the opposite face of the Holliday junction to RuvA and cleaves the junction symmetrically when specific sequences are present at the branch point (16 -19).…”

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Action of RuvAB at Replication Fork Structures

McGlynn¹,

Lloyd

2001

Journal of Biological Chemistry

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The replicative apparatus often encounters blocks to its progression that necessitate removal of the block and reloading of the replication machinery. In Escherichia coli, a major pathway of replication restart involves unwinding of the stalled fork to generate a four-stranded Holliday junction, which can then be cleaved by the RuvABC helicase-endonuclease. This fork regression may be catalyzed by RecG but is thought to occur even in its absence. Here we test whether RuvAB helicase can also catalyze the unwinding of forked DNA to form Holliday junctions. We find that fork DNA is unwound in the direction required for Holliday junction formation only if the loading of RuvB is restricted to the parental duplex DNA arm. If the binding of RuvB is unrestricted, then RuvAB preferentially unwinds forks in the opposite direction. This is probably related to the greater efficiency of two opposed RuvB hexamers operating across a junction compared with a single hexamer. These data argue against RuvAB acting directly at damaged replication forks and imply that other mechanisms must operate in vivo to catalyze Holliday junction formation.

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“…Electron microscopic visualization of the RuvAB-Holliday junction complex revealed that the junction lies sandwiched between two RuvA tetramers and that branch migration is driven by two oppositely oriented RuvB rings that bind to opposing arms (Parsons et al 1995;Yu et al 1997). During branch migration, the DNA passes through RuvA and exits the complex through the cavity in each RuvB ring (Hiom et al 1996).…”

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Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of Holliday junction resolution

Gool¹,

Hajibagheri²,

Stasiak³

et al. 1999

Genes & Development

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Genetic recombination can lead to the formation of intermediates in which DNA molecules are linked by Holliday junctions. Movement of a junction along DNA, by a process known as branch migration, leads to heteroduplex formation, whereas resolution of a junction completes the recombination process. Holliday junctions can be resolved in either of two ways, yielding products in which there has, or has not, been an exchange of flanking markers. The ratio of these products is thought to be determined by the frequency with which the two isomeric forms (conformers) of the Holliday junction are cleaved. Recent studies with enzymes that process Holliday junctions in Escherichia coli, the RuvABC proteins, however, indicate that protein binding causes the junction to adopt an open square-planar configuration. Within such a structure, DNA isomerization can have little role in determining the orientation of resolution. To determine the role that junction-specific protein assembly has in determining resolution bias, a defined in vitro system was developed in which we were able to direct the assembly of the RuvABC resolvasome. We found that the bias toward resolution in one orientation or the other was determined simply by the way in which the Ruv proteins were positioned on the junction. Additionally, we provide evidence that supports current models on RuvABC action in which Holliday junction resolution occurs as the resolvasome promotes branch migration.

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**The directionality of RuvAB‐mediated branch migration: in vitro studies with three‐armed junctions**

Cited by 19 publications

References 26 publications

The Role of RuvA Octamerization for RuvAB Function in Vitro and in Vivo

The Role of RuvA Octamerization for RuvAB Function in Vitro and in Vivo

Action of RuvAB at Replication Fork Structures

Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of Holliday junction resolution

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