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Holliday junction binding and processing by the RuvA protein of Mycoplasma pneumoniae
Stuart M Ingleston1, Mark J Dickman, Jane A Grasby
1Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham, UK.
Abstract:
The RuvA, RuvB and RuvC proteins of Escherichia coli act together to process Holliday junctions formed during recombination and DNA repair. RuvA has a well-defined DNA binding surface that is sculptured specifically to accommodate a Holliday junction and allow subsequent loading of RuvB and RuvC. A negatively charged pin projecting from the centre limits binding of linear duplex DNA. The amino-acid sequences forming the pin are highly conserved. However, in certain Mycoplasma and Ureaplasma species the structure is extended by four amino acids and two acidic residues forming a crucial charge barrier are missing. We investigated the significance of these differences by analysing RuvA from Mycoplasma pneumoniae. Gel retardation and surface plasmon resonance assays revealed that this protein binds Holliday junctions and other branched DNA structures in a manner similar to E. coli RuvA. Significantly, it binds duplex DNA more readily. However it does not support branch migration mediated by E. coli RuvB and when bound to junction DNA is unable to provide a platform for stable binding of E. coli RuvC. It also fails to restore radiation resistance to an E. coli ruvA mutant. The data presented suggest that the modified pin region retains the ability to promote junction-specific DNA binding, but acts as a physical obstacle to linear duplex DNA rather than as a charge barrier. They also indicate that such an obstacle may interfere with the binding of a resolvase. Mycoplasma species may therefore process Holliday junctions via uncoupled branch migration and resolution reactions.
Insights
Mycoplasma pneumoniae RuvA binds branched DNA like E. coli RuvA but more readily binds duplex DNA. This difference prevents it from supporting DNA repair processes, suggesting unique mechanisms in Mycoplasma species.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The RuvA, RuvB, and RuvC proteins in Escherichia coli are essential for processing Holliday junctions during DNA recombination and repair.
- RuvA possesses a specific DNA binding surface for Holliday junctions, facilitating RuvB and RuvC loading, with a central pin limiting linear DNA binding.
Purpose of the Study:
- To investigate the functional significance of structural differences in RuvA from Mycoplasma pneumoniae compared to E. coli RuvA.
- To determine how modifications in the RuvA pin region affect DNA binding and Holliday junction processing.
Main Methods:
- Gel retardation assays to assess DNA binding affinities.
- Surface plasmon resonance (SPR) to analyze protein-DNA interactions.
- Functional assays using an E. coli ruvA mutant to evaluate DNA repair restoration.
Main Results:
- Mycoplasma pneumoniae RuvA binds Holliday junctions and branched DNA structures similarly to E. coli RuvA.
- Mycoplasma pneumoniae RuvA exhibits increased binding to duplex DNA compared to E. coli RuvA.
- Mycoplasma pneumoniae RuvA does not support E. coli RuvB-mediated branch migration or stable E. coli RuvC binding and fails to restore radiation resistance in an E. coli ruvA mutant.
Conclusions:
- The modified pin region in Mycoplasma pneumoniae RuvA facilitates junction-specific binding but acts as a physical obstacle to linear DNA, unlike the charge barrier in E. coli RuvA.
- This structural difference may hinder resolvase binding, suggesting that Mycoplasma species might employ uncoupled branch migration and resolution pathways for Holliday junction processing.