Crystal structure of the phage T4 recombinase UvsX and its functional interaction with the T4 SF2 helicase UvsW

Abstract

Bacteriophage T4 provides an important model system for studying the mechanism of homologous recombination. We have determined the crystal structure of the T4 UvsX recombinase, and the overall architecture and fold closely resemble those of RecA, including a highly conserved ATP binding site. Based on this new structure, we reanalyzed electron microscopy reconstructions of UvsX-DNA filaments and docked the UvsX crystal structure into two different filament forms: a compressed filament generated in the presence of ADP and an elongated filament generated in the presence of ATP and aluminum fluoride. In these reconstructions, the ATP binding site sits at the protomer interface, as in the RecA filament crystal structure. However, the environment of the ATP binding site is altered in the two filament reconstructions, suggesting that nucleotide cannot be as easily accommodated at the protomer interface of the compressed filament. Finally, we show that the phage helicase UvsW completes the UvsX-promoted strand-exchange reaction, allowing the generation of a simple nicked circular product rather than complex networks of partially exchanged substrates.

Fig. 1

Comparison of the UvsX_30–358 monomer with RecA. (A) The structure of the UvsX_30–358 monomer. α-helices are shown in green, β-strands in purple and loops in grey. The N-terminal ATP-binding αβ core domain is oriented towards the right and the helical C-terminal domain is on the left. ATP is not present in the structure, but the phosphate group (orange/red ball-and-stick) occupies the pocket that accommodates the ATP β-phosphate. Loops L1 and L2 are not visible in the structure, but their locations are labeled together with the N- and C-termini. (B) The RecA monomer as determined by Chen and coworkers (PDB ID: 3CMX) shown in the same orientation as UvsX in (A) based on an alignment of the core domain secondary structures. The RecA structure includes loops L1 and L2 (labeled) and a bound ADP (shown in ball-and stick). Note that the N-terminal oligomerization α-helix that was removed in UvsX to facilitate crystallization is present in RecA. (C) Structure-based sequence alignment of UvsX and RecA derived from (A) and (B). Sequence numbering refers to UvsX. Cys316 in UvsX is highlighted in yellow; RecA Pro313 and UvsX Pro322 are marked cyan; other identical residues are marked with red boxes. The figure was prepared with ESPript. In (A), the semi-transparent yellow structure shows the predicted location of the C-terminal domain based on its location in RecA. The ~15° rotation between the observed and predicted positions occurs around a conserved Gly-Ile motif indicated by an asterisk in (A), (B) and (C).

Fig. 2

The dimeric arrangement of UvsX_30–358 in the crystal asymmetric unit. (A) UvsX_30–358 is colored as in Figure 1A. The ATP-binding sites are indicated by the phosphate groups (orange/red CPK), and are occluded at the dimer interface. The paired Cys316 residues at the dimer interface appear to form a partially occupied disulfide bridge (yellow CPK) in the crystal and in solution (see Figure 3). The positions of the N- and C-termini and Loops L1 and L2 are indicated. The positions of Pro322 highlighted in Figure 1C are marked with cyan asterisks. (B) Six dimers create one turn of a helical array in the P6₁ unit cell. Interactions between successive monomer A molecules (blue) mediate the helical filament with no contributions from monomer B (grey).

Fig. 3

SDS PAGE analysis of oxidized and reduced UvsX. (A) Wild type full length and truncated UvsX proteins show partial dimerization when oxidized and a single band when reduced. The N-terminal deletion mutant UvsX_30–391 shows degradation when oxidized, but the reduced sample runs as a single species. The C-terminal deletion mutant UvsX_1–358 and the core domain UvsX_30–358 migrate similarly on the gel. The band positions of protein standards are marked with their molecular weights (kDa) on the left. Note that the dimeric fractions of both samples which lack the disordered acidic C-terminal region run at a molecular weight more consistent with the calculated molecular weight. The calculated molecular weight values for each dimer are: 93 kDa for UvsX_1–391, 86 kDa for UvsX_30–391, 84 kDa for UvsX_1–358 and 80 kDa for UvsX_30–358. (B) The C316S point mutant of full-length UvsX fails to dimerize in oxidizing conditions. Wild type (wt) UvsX exhibits dimerization in the loading buffer without addition of oxidizing agents (right hand side). Reduced UvsX protein was supplemented with 5 mM TCEP, and oxidized protein with 1% H₂O₂.

Fig. 4

Electron microscopy of UvsX recombination filaments. (A) Reconstruction of the extended ‘active’ filament (grey) formed in the presence of dsDNA and ATP into which the UvsX crystal structure has been fitted (cyan). The C-terminal helical domain is pointing down towards the large groove. The filament has a rotation per subunit of 58.5° and axial rise per subunit of 16.1 Å. The 28 N-terminal residues of RecA were used to model the missing N-terminal UvsX residues (green ribbons). The positions of three residues in UvsX at the monomer-monomer interface that correspond to those in RecA involved in the ATP hydrolysis are shown as red (Lys246, Arg248), and yellow (Glu92) spheres. (B) The compressed ‘inactive’ filament formed in the presence of dsDNA and ADP in which the fitted UvsX structure is shown in dark blue. The filament has a rotation per subunit of 55.7° and axial rise per subunit of 10.8 Å. A bridge of density across the groove, corresponding to an interaction between residues 130–132 of one monomer and residues 285–288 of the other monomer, is indicated by red arrows.

Fig. 5

Monomer-monomer interactions in the RecA and UvsX active filaments. (A) RecA-RecA interaction derived from the structure of Chen and coworkers. ADP is bound at the interface and the residues involved in ATP hydrolysis, Lys248 (red), Lys250 (red) and Glu96 (yellow), are indicated. (B) The UvsX-UvsX interaction derived from the EM reconstruction. The ADP at the interface and the missing N-terminal α-helix (dark blue) are modeled from the RecA superposition shown in Figure 1B. By comparison with the RecA structure, residues Lys246 (red), Arg248 (red) and Glu92 (yellow), mediate ATP hydrolysis at the interface.

Fig. 6

UvsW allows completion of UvsX-promoted strand exchange. The migrations of nicked circular, single-strand circular and double-strand linear markers are indicated by arrows at the right (top to bottom, respectively). (A) Strand exchange reactions contained UvsX (1 µM), UvsY (0.5 µM), gp32 (2.5 µM), and the indicated concentration of UvsW, along with the DNA substrates (linear duplex DNA and homologous single-strand circles). Samples were removed at the following times: 2.5 min (lanes 2, 6, 10 and 14), 5 min (lanes 3, 7, 11 and 15), 10 min (lanes 4, 8, 12 and 16), and 22.5 min (lanes 5, 9, 13 and 17). The reaction in lane 1 contained UvsX, UvsY, and gp32 but no ATP. (B) UvsW allows completion of UvsX-promoted strand exchange in the absence of UvsY. Strand exchange reactions contained UvsX and gp32, each at 2.5 µM, the indicated concentration of UvsW, and no UvsY protein. Samples were removed at 5 min (lanes 1, 3, 5 and 7) and 22.5 min (lanes 2, 4, 6 and 8). (C) UvsW promotes strand exchange under conditions where UvsY is inhibitory. Strand exchange reactions contained UvsX, UvsY, and gp32, each at 2.5 µM, and the indicated concentration of UvsW. Samples were removed at 5 min (lanes 1, 3, 5 and 7) and 22.5 min (lanes 2, 4, 6 and 8). (D) Dda helicase and UvsW-K141R do not facilitate completion of UvsX-promoted strand exchange. Strand exchange reactions contained UvsX (2.5 µM), UvsY (0.5 µM), gp32 (2.5 µM), and, where indicated, UvsW (50 nM), UvsW-K141R (50 nM) or Dda (75 nM). Samples were removed at 5 min (lanes 1, 3, 5 and 7) and 25 min (lanes 2, 4, 6 and 8). The reaction in lane 9 was a control that contained UvsX, UvsY, and gp32, but no ATP.