Three New Structures of Left-Handed RadA Helical Filaments: Structural Flexibility of N-Terminal Domain Is Critical for Recombinase Activity

PLOS ONE, Mar 2009

RecA family proteins, including bacterial RecA, archaeal RadA, and eukaryotic Dmc1 and Rad51, mediate homologous recombination, a reaction essential for maintaining genome integrity. In the presence of ATP, these proteins bind a single-strand DNA to form a right-handed nucleoprotein filament, which catalyzes pairing and strand exchange with a homologous double-stranded DNA (dsDNA), by as-yet unknown mechanisms. We recently reported a structure of RadA left-handed helical filament, and here present three new structures of RadA left-handed helical filaments. Comparative structural analysis between different RadA/Rad51 helical filaments reveals that the N-terminal domain (NTD) of RadA/Rad51, implicated in dsDNA binding, is highly flexible. We identify a hinge region between NTD and polymerization motif as responsible for rigid body movement of NTD. Mutant analysis further confirms that structural flexibility of NTD is essential for RadA's recombinase activity. These results support our previous hypothesis that ATP-dependent axial rotation of RadA nucleoprotein helical filament promotes homologous recombination.

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Three New Structures of Left-Handed RadA Helical Filaments: Structural Flexibility of N-Terminal Domain Is Critical for Recombinase Activity

et al. (2009) Three New Structures of Left-Handed RadA Helical Filaments: Structural Flexibility of N- Terminal Domain Is Critical for Recombinase Activity. PLoS ONE 4(3): e4890. doi:10.1371/journal.pone.0004890 Three New Structures of Left-Handed RadA Helical Filaments: Structural Flexibility of N-Terminal Domain Is Critical for Recombinase Activity Yu-Wei Chang 0 Tzu-Ping Ko 0 Chien-Der Lee 0 Yuan-Chih Chang 0 Kuei-Ann Lin 0 Chia-Seng Chang 0 Andrew H.-J. Wang 0 Ting-Fang Wang 0 Eshel Ben-Jacob, Tel Aviv University, Israel 0 1 Institute of Biochemical Science, National Taiwan University , Taipei, Taiwan , 2 Institute of Biological Chemistry , Academia Sinica, Taipei, Taiwan , 3 Institute of Physics , Academia Sinica, Taipei, Taiwan , 4 Institute of Molecular Biology, Academia Sinica , Taipei , Taiwan RecA family proteins, including bacterial RecA, archaeal RadA, and eukaryotic Dmc1 and Rad51, mediate homologous recombination, a reaction essential for maintaining genome integrity. In the presence of ATP, these proteins bind a singlestrand DNA to form a right-handed nucleoprotein filament, which catalyzes pairing and strand exchange with a homologous double-stranded DNA (dsDNA), by as-yet unknown mechanisms. We recently reported a structure of RadA lefthanded helical filament, and here present three new structures of RadA left-handed helical filaments. Comparative structural analysis between different RadA/Rad51 helical filaments reveals that the N-terminal domain (NTD) of RadA/Rad51, implicated in dsDNA binding, is highly flexible. We identify a hinge region between NTD and polymerization motif as responsible for rigid body movement of NTD. Mutant analysis further confirms that structural flexibility of NTD is essential for RadA's recombinase activity. These results support our previous hypothesis that ATP-dependent axial rotation of RadA nucleoprotein helical filament promotes homologous recombination. - Competing Interests: The authors have declared that no competing interests exist. Homologous recombination is a ubiquitous mechanism for maintaining genome integrity and also for generating genetic diversity in sexual reproductive organisms. This reaction is catalyzed by RecA family proteins, including bacterial RecA, archaeal RadA, and eukaryal Rad51 and Dmc1. The current model holds that, in the presence of ATP, the recombinases coat a primary single-stranded DNA (ssDNA) to form a nucleoprotein right-handed helical filament, and initiate a search for a secondary homologous stretches of double-stranded DNA (dsNDA). The ssDNA then invades and displaces the homologous strand in the donor dsDNA, resulting in a new heteroduplex (or D-loop). Eventually, the homologous ssDNA will be expelled from the nucleoprotein filament [1,2,3]. Escherichia coli RecA (EcRecA) is the founding member of the RecA protein family. It contains three major structural domains: a small N-terminal domain (NTD), a catalytic domain (CAD) and a large C-terminal domain (CTD). The CAD, often referred to as the RecA fold [4], is structurally similar to the ATPase domains of DNA/RNA helicases, F1 ATPases, chaperone-like ATPases, and membrane transporters [5]. The CAD contains two disordered loops (the L1 and L2 motifs) that bind to ssDNA and are responsible for the ssDNA-stimulated ATPase activity [6]. Two positively-charged CAD residues, Arg243 and Lys245, are responsible for binding to donor dsDNA [7,8,9,10]. The CTD may also have a similar function in the RecA-ssDNA nucleoprotein filament of capturing donor dsDNA [7,8]. RecA polymerization is mediated by the polymerization motif (PM) that is located between the NTD and the CAD. PM contains a hydrophobic residue (i.e., Ile26) that docks within the hydrophobic pocket of the neighboring CAD. Nikola Pavletich and colleagues recently reported the crystal structures of EcRecA-ssDNA and EcRecAdsDNA nucleoprotein complexes with Mg2+, ADP and AlF42 [11]. These right-handed filament structures have provided unprecedented new insights into the mechanisms and energetic of EcRecA. [11,12]. Here, ADP-AlF42 was used to mimic the ADP-Pi, because AlF42 is able to substitute for inorganic phosphate (Pi) after the hydrolysis of ATP. The EcRecA-ssDNAMg2+-ADP-AlF42 nucleoprotein filament represents the structural intermediate responsible for homology pairing to a donor dsDNA. By contrast, the RecA-dsDNA-ADP-AlF42-Mg2+ crystal structure was postulated to be an end product after strand exchange reaction between RecA-ssDNA nucleoprotein filament and a homologous dsDNA target, implying that RecA protein filaments may complete all functions (including ssDNA binding, donor dsDNA capturing and strand exchange) within the axes of righthanded filaments. Here we consider an alternative possibility that the RecA-dsDNA crystal structures might simply represent annealing products of the ssDNA in RecA-ssDNA nucleoprotein filament and a complementary ssDNA. Firstly, in the RecAssDNA filament structure, the purine a (...truncated)


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Yu-Wei Chang, Tzu-Ping Ko, Chien-Der Lee, Yuan-Chih Chang, Kuei-Ann Lin, Chia-Seng Chang, Andrew H.-J. Wang, Ting-Fang Wang. Three New Structures of Left-Handed RadA Helical Filaments: Structural Flexibility of N-Terminal Domain Is Critical for Recombinase Activity, PLOS ONE, 2009, Volume 4, Issue 3, DOI: 10.1371/journal.pone.0004890