Fig 1.
ATPase profile from EcRecA and HsRecA in the presence and absence of SSB protein.
(A) Reaction 1: contained 5 μM nt M13mp18 cssDNA and 3 μM HsRecA or EcRecA, were previously incubated per 20 min at 37°C, following, 3 μM ATP and 0.5 μM SSB. Reaction 2: reaction 1 without SSB protein addition. Time 0 min indicates the addition of ATP and SSB. (B) The reaction contained 5 μM nt M13mp18 cssDNA, 3 μM ATP and 0.35 μM SSB, were previously incubated per 10 min at 37°C, following addition of 3 μM HsRecA or EcRecA (time 0 min).
Fig 2.
DNA strand exchange promoted by the wild-type HsRecA and EcRecA proteins.
(A) The three DNA strand exchange containing 10 μM nt M13mp18 cssDNA and 3.5 μM HsRecA or EcRecA, were previously incubated per 20 min at 37°C, and then 3 μM ATP and 1 μM SSB were added and incubated for an additional 10 min. The minutes shown represents the time of reaction after addition of 20 μM nt M13mp18 ldsDNA. (B) The percentage of duplex substrate converted into the nicked circular duplex (NC product) is plotted against the time.
Fig 3.
Electronic microscopy of EcRecA and HsRecA filaments in the absence or presence of EcSSB.
(A) EcRecA-cssDNA filaments without EcSSB, (B) HsRecA-cssDNA filaments without EcSSB, (C) EcRecA-cssDNA filaments with EcSSB, (D) HsRecA-cssDNA filament with EcSSB, (E) Avarege length of 10 HsRecA and EcRecA filaments in the conditions assayed. Letters above each bar refer back to panels A-D. Reactions containing 6.7 μM HsRecA or EcRecA and 20 μM M13mp18 cssDNA were incubated at 37°C for 20 min, with or without 2 μM Ec SSB. After 10 min, filaments were stabilized by a 3 min incubation with 3 μM ATPγS and spread on an Alcian grid.
Fig 4.
Ribbon diagram of the monomeric crystal structure of HsRecA protein.
Regions that could be modeled were indicated by the last residue-number. HsRecA protein is composed of N-terminal domain (NTD), a central core ATPase domain and a large C-terminal domain (CTD). The core ATPase domain contains one Ca2+ ion (magenta sphere), coordinated by Asn119 and Asp120 and the ATPase activity site is partially occupied by ATP and ADP. Figure was prepared using the STRIDE program for secondary structure assignment [55], and visualized using PyMOL [56].
Table 1.
Data collection and refinement statistics.
Fig 5.
Omit maps (mFo-DFc) at the ATP binding site: (A) Refinement with ATP at 100% occupancy; the negative electron density over the γ-phosphate indicates that it should not be at this full occupancy. (B) Refinement with ATP at 39% occupancy; the positive electron density over the corresponding ADP moiety indicates that it should be at full occupancy. The omit maps are contoured at +3 (green) and -3 (red) σ levels.
Fig 6.
3D structural and amino acid sequence alignment from H. seropedicae (HsRecA), E. coli (EcRecA), M. tuberculosis (MtRecA), M. smegmatis (MsRecA), D. radiodurans (DrRecA), N. gonorrhoeae (NgRecA), P. aeruginosa (PaRecA), M. voltae (MvRadA), P. furiosus (RadA), H. sapiens (HsRad51), P. furiosus (Rad51), S. cerevisiae (ScRad51 and Dmc1).
Part of the N-terminus has been removed for clarity. The position of the core ATPase domain and the C-terminus are indicated. Functional motifs are indicated above their corresponding amino acid sequences: N-PM and core-PM, N-terminal and core polymerization motif, respectively, the ATP binding Walker A and B motifs, the putative DNA binding sites Loop L1 and L2. The secondary structure of HsRecA and HsDmc1 are indicated at the top and bottom, respectively. The 3D structural and amino acid alignment was performed using the MultiProt and T-Coffee programs [61,62]. The results are visualized using ESPript—http://espript.ibcp.fr [63].
Table 2.
Summary of hydrogen bonds formed between the N-PM and the Core-PM in RecA protein structures.
The residues and their atoms involved in the interaction are indicated, as well as the atomic distances. Interactions were determined using the CCP4 application (Protein Interfaces, Surfaces and Assemblies—PISA) [52,64,65].
Fig 7.
Superposition inactive and presynaptic monomers of EcRecA protein over the inactive HsRecA monomer.
EcRecA protein is colored in gray and HsRecA in cyan. (A) Superposition of the inactive EcRecA structure (pdb entry 1XMV) over the HsRecA structure. Both structures have a β-loop motif in the N-PM. In the EcRecA structure, a Mg2+ ion interacts with the N-PM. In the HsRecA structure, a Ca2+ ion interacts with the Core-PM. (B) Superposition of presynaptic monomers of EcRecA structure (pdb entry 3CMU:A) over the HsRecA structure. The N-PM in the active EcRecA structure assumes a β-strand motif. The nucleotides ADP and ADP-AlF4, Mg2+ and Ca2+ ions, and the ssDNA bound in the EcRecA structure was removed for clarity. Figure was prepared using the STRIDE program for secondary structure assignment [55]. The superposition was generated using the MultiProt program [61] and visualized using PyMOL [56].
Fig 8.
Electrostatic potential distribution on the solvent-accessible surface of HsRecA protein structure.
(A) Side view of two subsequent helical hexamers. (B) and (C), 5′ and 3′ views, relative to the axial direction of the filament, respectively. The surface potential representation has charge levels from -3kT/e (red) to +3kT/e (blue). The electrostatic potential distribution was generated using the APBS program, side chain atom not ordered in the crystal were added using the PDB2PQR program and protonation states at pH 7.5 were assigned with the PROPKA program [67–69], and visualized using PyMOL [56].
Fig 9.
Ribbon diagram of two subsequent helical hexamers of HsRecA protein.
Each monomer is colored differently. The hexameric structure has 61-fold symmetry, the helical filament has a pitch of 91.3 Å. Figures were prepared using the STRIDE program for secondary structure assignment [55], and visualized using PyMOL [56].