Structural and functional analyses of five conserved positively charged residues in the L1 and N-terminal DNA binding motifs of archaeal RADA protein - PubMed (original) (raw)
Structural and functional analyses of five conserved positively charged residues in the L1 and N-terminal DNA binding motifs of archaeal RADA protein
Li-Tzu Chen et al. PLoS One. 2007.
Abstract
RecA family proteins engage in an ATP-dependent DNA strand exchange reaction that includes a ssDNA nucleoprotein helical filament and a homologous dsDNA sequence. In spite of more than 20 years of efforts, the molecular mechanism of homology pairing and strand exchange is still not fully understood. Here we report a crystal structure of Sulfolobus solfataricus RadA overwound right-handed filament with three monomers per helical pitch. This structure reveals conformational details of the first ssDNA binding disordered loop (denoted L1 motif) and the dsDNA binding N-terminal domain (NTD). L1 and NTD together form an outwardly open palm structure on the outer surface of the helical filament. Inside this palm structure, five conserved basic amino acid residues (K27, K60, R117, R223 and R229) surround a 25 A pocket that is wide enough to accommodate anionic ssDNA, dsDNA or both. Biochemical analyses demonstrate that these five positively charged residues are essential for DNA binding and for RadA-catalyzed D-loop formation. We suggest that the overwound right-handed RadA filament represents a functional conformation in the homology search and pairing reaction. A new structural model is proposed for the homologous interactions between a RadA-ssDNA nucleoprotein filament and its dsDNA target.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Crystal packing and quaternary structures.
(A) _Sso_RadA protomers packed into three extended helical filaments. Chain A was located at the origin of the unit cell, whereas chains B and C were located one-third and two-third diagonal to the unit cell. (B) Side view of the Sso_RadA right-handed helical filament crystal structure. The helical pitch of the filament is 98 Å. Each protomer is shown in a different color. The N-terminal domain (NTD), polymerization motif (PM), and central ATPase domain are indicated. (C) The Phe73 of the PM is buried in the hydrophobic pocket of the neighboring ATPase domain. Several hydrophobic residues that interact with the Phe73 side chain are indicated. The interactions result in the assembly of Sso_RadA protomers into a filament. 2_F o–_F c electron density maps (contoured at 1.0 σ), corresponding to the PM are shown in orange.
Figure 2. Structure of the L1 motif.
(A) Sequence alignment of RadA homologs from S. solfataricus (Sso) RadA, M. voltae (Mv) RadA, P. furiosus (Pf) Rad51, H. sapiens (Hs) Rad51 and Dmc1, and S. cerevisiae (Sc) Rad51 and Dmc1. Three conserved arginine residues are shown in cyan. (B) A ribbon diagram of the L1 motif showing two alpha-helices (grey). The hinge region is depicted with a ball-and-stick model (yellow). The side chains of three conserved arginine residues are shown in green. 2_F_ o–F c electron density maps (contoured at 1.0σ), corresponding to the ssDNA binging site, are shown in cyan. (C) Surface charge potential of the L1 motif. The positively and negatively charged regions are indicated in blue and red, respectively. The linear basic patch is ∼18 Å in length.
Figure 3. Structure of the N-terminal domain (NTD).
(A) Sequence alignment of the NTD and (HhH)2 domains in RadA homologs from S. solfataricus(Sso) RadA), M. voltae (Mv) RadA, P. furiosus (Pf) Rad51, H. sapiens(Hs) Rad51 and Dmc1, and S. cerevisiae (Sc) Rad51 and Dmc1. The first and second HhH motifs are denoted as H1'h'H2' and H1hH2, respectively. (B) Cartoon diagram of the (HhH)2 domains. The first and second HhH motifs are connected by the connector alpha-helix Hc (yellow). (C) A ribbon diagram of the NTD. The hinge regions (h' and h) are depicted with a ball-and-stick model (green). 2_F_ o–F c electron density maps (contoured at 1.0 σ) of several key amino acid residues are in purple. Oxygen and nitrogen atoms are shown in red and blue, respectively. (D) Surface charge potential of the NTD. The positively and negatively charged regions are indicated by blue and red, respectively. The two borders of this 92° arched basic patch are 15 Å and 14 Å in length, respectively. (E) Top view of the NTD surface charge potential reveals a central channel that is 18 Å long and 14 Å wide. The channel is too narrow to accommodate a B-type dsDNA substrate.
Figure 4. Spatial arrangement of the L1 motif and the NTD along the 31 overwound right-handed _Sso_RadA filament.
(A) Quaternary structure. The putative dsDNA binding regions in the NTD are shown in blue. The L1 and L2 ssDNA binding motifs are shown in pink and green, respectively. ATP binding sites are shown in yellow. The polymerization motif (PM) is indicated by an arrow. (B) A local surface charge potential of the L1 motif and the NTD region. Positive and negative charges are indicated by blue and red, respectively. (C) A ribbon diagram of two neighboring protomers (grey) showing the L1 motif (pink) and the NTD (cyan). The side chains of key basic residues K27, K60, R217, R223, and R229 are depicted in ball-and-stick representations.
Figure 5. D-loop formation.
_Sso_RadA-promoted homologous strand assimilation between a γ-32P labeled oligonucleotide and a dsDNA plasmid was carried out as described previously ,
Figure 6. ssDNA binding.
The 5′-biotinylated (dT)50 oligonucleotide (10 µM in nucleotides) was first injected into BIAcore SA sensor chips. Wild-type or point mutant _Sso_RadA protein (1, 5, 10 µM) was passed over the chip at 25°C. Curves represent responses with the background subtracted. Binding signal was not detected when solutions that did not contain _Sso_RadA protein were injected.
Figure 7. dsDNA binding.
Nucleoprotein gel of reactions containing 4.3 µM (in bps) dsDNA without _Sso_RadA protein (first lane in upper panel) or with 1, 5, or 10 µM of _Sso_RadA protein. The nucleoprotein complexes were fixed with glutaraldehyde to a final concentration of 2.5%, separated from free DNA on a 0.5% agarose gel, and visualized with ethidium bromide.
Figure 8. A new hypothesis for homology interactions mediated by RadA protein filaments.
Interactions between three arginine residues of the L1 motif and sugar-phosphate backbone of ssDNA result in the nucleotide bases of ssDNA facing the NTD (Figure 1). An anionic dsDNA associates with the NTD along the border of a 92° basic arch via electrostatic interactions or hydrogen bonding. Lys27 and Lys60 are located at each end of this arched basic patch (Figure 3). As a result, NTD-dsDNA association may lead to DNA bending or distortion or flipping of base pairs. L1-ssDNA and NTD-dsDNA interactions function in unison to mediate homologous search and pairing between a 31 overwound right-handed RadA-ssDNA nucleoprotein filament and dsDNA.
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