Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

Homologous recombination (HR), a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR) processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR). T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms

Insights into the mechanism of Rad51 recombinase from the structure and properties of a filament interface mutant

Rad51 protein promotes homologous recombination in eukaryotes. Recombination activities are activated by Rad51 filament assembly on ssDNA. Previous studies of yeast Rad51 showed that His352 occupies an important position at the filament interface, where it could relay signals between subunits and active sites. To investigate, we characterized yeast Rad51 H352A and H352Y mutants, and solved the structure of H352Y. H352A forms catalytically competent but salt-labile complexes on ssDNA. In contrast, H352Y forms salt-resistant complexes on ssDNA, but is defective in nucleotide exchange, RPA displacement and strand exchange with full-length DNA substrates. The 2.5 Å crystal structure of H352Y reveals a right-handed helical filament in a high-pitch (130 Å) conformation with P61 symmetry. The catalytic core and dimer interface regions of H352Y closely resemble those of DNA-bound Escherichia coli RecA protein. The H352Y mutation stabilizes Phe187 from the adjacent subunit in a position that interferes with the γ-phosphate-binding site of the Walker A motif/P-loop, potentially explaining the limited catalysis observed. Comparison of Rad51 H352Y, RecA–DNA and related structures reveals that the presence of bound DNA correlates with the isomerization of a conserved cis peptide near Walker B to the trans configuration, which appears to prime the catalytic glutamate residue for ATP hydrolysis

Coordinated Binding of Single-Stranded and Double-Stranded DNA by UvsX Recombinase

<div><p>Homologous recombination is important for the error-free repair of DNA double-strand breaks and for replication fork restart. Recombinases of the RecA/Rad51 family perform the central catalytic role in this process. UvsX recombinase is the RecA/Rad51 ortholog of bacteriophage T4. UvsX and other recombinases form presynaptic filaments on ssDNA that are activated to search for homology in dsDNA and to perform DNA strand exchange. To effectively initiate recombination, UvsX must find and bind to ssDNA within an excess of dsDNA. Here we examine the binding of UvsX to ssDNA and dsDNA in the presence and absence of nucleotide cofactor, ATP. We also examine how the binding of one DNA substrate is affected by simultaneous binding of the other to determine how UvsX might selectively assemble on ssDNA. We show that the two DNA binding sites of UvsX are regulated by the nucleotide cofactor ATP and are coordinated with each other such that in the presence of ssDNA, dsDNA binding is significantly reduced and correlated with its homology to the ssDNA bound to the enzyme. UvsX has high affinity for dsDNA in the absence of ssDNA, which may allow for sequestration of the enzyme in an inactive form prior to ssDNA generation.</p></div

Binding of UvsX to dsDNA in the presence of excess homologous or heterologous ssDNA.

<p>Various amounts of UvsX were added to mixtures containing final concentrations of 2 µM dsDNA (dT₂₅:dA₂₂XA₂ oligo 2: oligo 1), 2.5 mM ATP, plus 40 µM of either homologous (dA_25, oligo 4) or heterologous (dC₂₅ oligo 3) ssDNA. The amplitude of fluorescence quenching was graphed as a function of UvsX concentration. Data for homologous ssDNA were fit to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066654#pone.0066654.e001" target="_blank">Equation 1</a> to determine an apparent <i>K_d</i> value (black line). Data for heterologous ssDNA were fit to a line (gray line).</p

Apparent dissociation constants for UvsX binding to dsDNA or ssDNA^a.

a<p>Equilibrium binding data derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066654#pone-0066654-g002" target="_blank">Figures 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066654#pone-0066654-g003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066654#pone-0066654-g004" target="_blank">4</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066654#pone-0066654-g006" target="_blank">6</a>.</p>b<p>No binding observed under the experimental conditions used.</p>c<p>Not Determined. Measuring the apparent <i>K_d</i> for dsDNA in the presence of ATP and heterologous ssDNA requires unattainably high protein concentrations.</p>d<p>Based on observation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066654#pone-0066654-g004" target="_blank">Figure 4</a> that homologous dsDNA does not destabilize UvsX-ssDNA interactions.</p

Binding of UvsX to ssDNA in the presence of increasing amounts of homologous dsDNA 3 µM UvsX (final concentration) was added to mixtures containing final concentrations of 2 µM ssDNA (dA₂₂XA₂, oligo 1), 2.5 mM ATP, and various concentrations of homologous dsDNA (dT₂₅:dA₂₅, oligo 2:oligo 4).

<p>The amplitude of fluorescence quenching was graphed as a function dsDNA and a line was fit (solid line) to demonstrate the trend.</p

Binding of UvsX to dsDNA in the presence of increasing amounts of homologous ssDNA.

<p>A) 2 µM UvsX (final concentration) was added to a mixture of 2 µM dsDNA (dT₂₅:dA₂₂XA₂ oligo 2:oligo 1), 2.5 mM ATP, and various amounts of homologous ssDNA (dA_25, oligo 4). The amplitude of fluorescence quenching was graphed as a function of ssDNA concentration and fit to a single exponential function (solid line) to demonstrate the trend. B) Schematic of strand exchange reaction used to determine if strand exchange is occurring during the monitoring of binding reactions depicted in A. C) Strand exchange reaction containing 0 or 2 µM UvsX, 8 µM ssDNA (dA₂₅) and 2 µM ³²-P labeled dsDNA (dA₂₅:dT₂₅) in the presence on 2.5 mM ATP. Control lane “C” indicated where on the gel the outgoing strand would be seen if strand exchange were to occur.</p

Binding of UvsX to double-stranded DNA in the absence or presence of nucleotide cofactors.

<p>Reactions were initiated by the addition of UvsX (final concentrations indicated on graph) to dsDNA, oligonucleotide dT₂₅:dA₂₂XA₂ (oligo 2:oligo 1) and nucleotide cofactors final concentrations are indicated below. The amplitude of fluorescence quenching was graphed as a function of UvsX concentrations and fit to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066654#pone.0066654.e001" target="_blank">Equation 1</a> to determine an apparent <i>K_d</i> value. (A) 0.5 µM (nucleotide pairs) dsDNA (B) 2 µM dsDNA and 2.5 mM ATP (C) 2 µM dsDNA and 900 µM ATPγS.</p

Effects of UvsX protein on fluorescence emission spectra of AlexaFluor 546-labeled oligonucleotides.

<p>Conditions were as described in Materials and Methods. The fluorescence emission spectra of (A) 2 µM (nucleotide pairs) double-stranded oligonucleotide dT₂₅:dA₂₂XA₂ (oligo 2:oligo 1), and (B) 2 µM (nucleotides) single-stranded oligonucleotide dA₂₂XA₂ (oligo 1), were recorded in the absence of UvsX (solid line), in the presence of a saturating amount of UvsX (2 µM) (dashed line), and in the presence of saturating amounts of both UvsX and ATP (2.5 mM) (dotted line).</p