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Structural basis for inhibition of homologous recombination by the RecX protein.

Ragone S, Maman JD, Furnham N, Pellegrini L - EMBO J. (2008)

Bottom Line: The relative arrangement of the repeats generates an elongated and curved shape that is well suited for binding within the helical groove of the RecA filament.Structure-based mutagenesis confirms that conserved basic residues on the concave side of RecX are important for repression of RecA activity.Analysis of RecA filament dynamics in the presence of RecX shows that RecX actively promotes filament disassembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.

ABSTRACT
The RecA/RAD51 nucleoprotein filament is central to the reaction of homologous recombination (HR). Filament activity must be tightly regulated in vivo as unrestrained HR can cause genomic instability. Our mechanistic understanding of HR is restricted by lack of structural information about the regulatory proteins that control filament activity. Here, we describe a structural and functional analysis of the HR inhibitor protein RecX and its mode of interaction with the RecA filament. RecX is a modular protein assembled of repeated three-helix motifs. The relative arrangement of the repeats generates an elongated and curved shape that is well suited for binding within the helical groove of the RecA filament. Structure-based mutagenesis confirms that conserved basic residues on the concave side of RecX are important for repression of RecA activity. Analysis of RecA filament dynamics in the presence of RecX shows that RecX actively promotes filament disassembly. Collectively, our data support a model in which RecX binding to the helical groove of the filament causes local dissociation of RecA protomers, leading to filament destabilisation and HR inhibition.

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Model of RecX-dependent destabilisation of RecA nucleoprotein filaments. RecX binding to the filament causes localised dissociation of RecA protomers and increased number of 5′-ends, leading to overall filament disassembly.
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f7: Model of RecX-dependent destabilisation of RecA nucleoprotein filaments. RecX binding to the filament causes localised dissociation of RecA protomers and increased number of 5′-ends, leading to overall filament disassembly.

Mentions: The structural evidence presented here does not support a mode of RecX binding that involves the specific recognition of the extremities of the RecA filament. In addition, the SPR analysis of the effect of RecX on the dynamic behaviour of RecA filaments shows that RecX actively promotes the dissociation of RecA from the filament. Accordingly, a satisfactory model of RecX's role in HR should account for its ability to bind anywhere along the filament and for its active role in promoting filament disassembly. Clearly, current mechanistic proposals of RecX function (VanLoock et al, 2003; Drees et al, 2004a) need to be integrated in a new model that can explain all the evidence now available. Thermodynamic considerations predict that filament disassembly does not ensue by dissociation of RecA protomers from internal sites of the filament (McGhee and Von Hippel, 1974; Schwarz and Watanabe, 1983). Indeed, single-molecule fluorescence assays demonstrated that RecA protomers dissociate preferentially from filament ends (Joo et al, 2006). Thus, to promote filament disassembly RecX must either enhance the rate of RecA dissociation per filament end or increase the actual number of ends. We favour the latter proposition as it accounts for RecX's ability to bind anywhere along the filament. We propose that binding of RecX within the helical groove of the nucleoprotein filament might facilitate the removal of one or more RecA protomers, thus generating a discontinuity in the RecA coating of the DNA. Dissociation of RecA would then commence from the newly generated 5′-end, whereas the continuing association of RecX near the newly created 3′-end would prevent further addition of RecA protomers. Thus, the extent of RecA dissociation would be proportional to RecX concentration: the more RecX molecules bind to the filament, the more filament interruptions would occur, therefore increasing the overall rate of filament disassembly (Figure 7).


Structural basis for inhibition of homologous recombination by the RecX protein.

Ragone S, Maman JD, Furnham N, Pellegrini L - EMBO J. (2008)

Model of RecX-dependent destabilisation of RecA nucleoprotein filaments. RecX binding to the filament causes localised dissociation of RecA protomers and increased number of 5′-ends, leading to overall filament disassembly.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2500204&req=5

f7: Model of RecX-dependent destabilisation of RecA nucleoprotein filaments. RecX binding to the filament causes localised dissociation of RecA protomers and increased number of 5′-ends, leading to overall filament disassembly.
Mentions: The structural evidence presented here does not support a mode of RecX binding that involves the specific recognition of the extremities of the RecA filament. In addition, the SPR analysis of the effect of RecX on the dynamic behaviour of RecA filaments shows that RecX actively promotes the dissociation of RecA from the filament. Accordingly, a satisfactory model of RecX's role in HR should account for its ability to bind anywhere along the filament and for its active role in promoting filament disassembly. Clearly, current mechanistic proposals of RecX function (VanLoock et al, 2003; Drees et al, 2004a) need to be integrated in a new model that can explain all the evidence now available. Thermodynamic considerations predict that filament disassembly does not ensue by dissociation of RecA protomers from internal sites of the filament (McGhee and Von Hippel, 1974; Schwarz and Watanabe, 1983). Indeed, single-molecule fluorescence assays demonstrated that RecA protomers dissociate preferentially from filament ends (Joo et al, 2006). Thus, to promote filament disassembly RecX must either enhance the rate of RecA dissociation per filament end or increase the actual number of ends. We favour the latter proposition as it accounts for RecX's ability to bind anywhere along the filament. We propose that binding of RecX within the helical groove of the nucleoprotein filament might facilitate the removal of one or more RecA protomers, thus generating a discontinuity in the RecA coating of the DNA. Dissociation of RecA would then commence from the newly generated 5′-end, whereas the continuing association of RecX near the newly created 3′-end would prevent further addition of RecA protomers. Thus, the extent of RecA dissociation would be proportional to RecX concentration: the more RecX molecules bind to the filament, the more filament interruptions would occur, therefore increasing the overall rate of filament disassembly (Figure 7).

Bottom Line: The relative arrangement of the repeats generates an elongated and curved shape that is well suited for binding within the helical groove of the RecA filament.Structure-based mutagenesis confirms that conserved basic residues on the concave side of RecX are important for repression of RecA activity.Analysis of RecA filament dynamics in the presence of RecX shows that RecX actively promotes filament disassembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, UK.

ABSTRACT
The RecA/RAD51 nucleoprotein filament is central to the reaction of homologous recombination (HR). Filament activity must be tightly regulated in vivo as unrestrained HR can cause genomic instability. Our mechanistic understanding of HR is restricted by lack of structural information about the regulatory proteins that control filament activity. Here, we describe a structural and functional analysis of the HR inhibitor protein RecX and its mode of interaction with the RecA filament. RecX is a modular protein assembled of repeated three-helix motifs. The relative arrangement of the repeats generates an elongated and curved shape that is well suited for binding within the helical groove of the RecA filament. Structure-based mutagenesis confirms that conserved basic residues on the concave side of RecX are important for repression of RecA activity. Analysis of RecA filament dynamics in the presence of RecX shows that RecX actively promotes filament disassembly. Collectively, our data support a model in which RecX binding to the helical groove of the filament causes local dissociation of RecA protomers, leading to filament destabilisation and HR inhibition.

Show MeSH
Related in: MedlinePlus