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.
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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)