Figure 1.
Cation-induced conformational rearragement of conserved amino acid residues of RadA.
(A) Subunit interface of MvRadA (MvRAD51) structure in the absence of potassium cation (PDB code 1T4G). (B) Subunit interface region of MvRAD51 structure in the presence of potassium cation (PDB code 1XU4). Structural figures were generated using Pymol. (C) Sequence alignment of WalkerA/P-loop and L2 ssDNA binding region of H. sapiens (Hs), S. cerevisiae (Sc) and M. voltae (Mv) recombinases. HsRAD51 residues F129 and H294 are indicated with asterisks.
Figure 2.
Mutation of HsRAD51(F129) and HsRAD51(H294) residues affect ATP turnover.
(A) Purification of wild type and HsRAD51 substitution mutant proteins. Protein (1 µg) analyzed by 12% SDS-PAGE. (B) Steady-state ATPase activity with ssDNA in the presence of 150 mM KCl. (C) ATPγS binding by wild type and HsRAD51 substitution mutant proteins in the presence of ssDNA and 150 mM KCl. (D) ADP binding by wild type and HsRAD51 substitution mutant proteins in the presence of ssDNA and 150 mM KCl. (E) ATP turnover (kcat) with ssDNA and dsDNA in the presence and absence of KCl (K+). kcat values were calculated by Michaelis-Menten analysis. Error bars indicate standard deviation from at least three independent experiments.
Table 1.
Summary of ATP hydrolysis and nucleotide binding data of HsRAD51 wild type and HsRAD51(F129V) and HsRAD51(H294V) mutant proteins.
Figure 3.
HsRAD51(F129V) and HsRAD51(H294V) are deficient in D-loop formation and strand exchange.
(A) In vitro D-loop assay reaction schematic. (B) 0.8 µM of HsRAD51, HsRAD51(F129V), or HsRAD51(H294V) and [P32]-labeled ssDNA (90mer; 2.4 µM nt) were preincubated for 10 min at 37°C in the reaction buffer containing 1 mM ATP and 1 mM MgCl2 or CaCl2. Reactions were initiated by the addition of supercoiled pBS SK(-) plasmid DNA (35 µM bp). After 15 min, reactions were terminated by the addition of proteinase-K and SDS. Joint molecules (JMs) were analyzed on a 0.9% agarose gel. (C) Analysis of salt and RPA requirement for strand exchange. Reaction schematic shown above: HsRAD51 (5 µM) and φX174 circular ssDNA (30 µM nt) were pre-incubated with 2.5 mM ATP and 1 mM MgCl2 at 37°C for 5 m prior to the addition of 150 mM NaNH4HPO4 (if indicated) and linear φX174 dsDNA (15 µM bp). After 5 m, HsRPA (2 µM) was added (if indicated) and the incubation was continued for 3 h. Samples were deproteinized and analyzed on 0.9% agarose gel with 0.1 µg/mL ethidium bromide.
Figure 4.
F129 and H294 of HsRAD51 are critical for DNA binding in the presence of ATP.
(A) ssDNA binding analysis of wild type and HsRAD51 substitution mutant proteins by surface Plasmon resonance (SPR, Biacore) in the absence of an adenine nucleotide, in the presence of ADP and in the presence of ATP. Association and dissociation curve corresponding to 800 nM of each protein is shown. (B) dsDNA binding analysis of wild type and HsRAD51 substitution mutant proteins.
Table 2.
Summary of DNA binding data of HsRAD51 wild type and HsRAD51(F129V) and HsRAD51(H294V) mutant proteins.*