A RecA mutant, RecA(730), suppresses the recombination deficiency of the RecBC(1004)D-chi* interaction in vitro and in vivo

Abstract

In Escherichia coli, homologous recombination initiated at double-stranded DNA breaks requires the RecBCD enzyme, a multifunctional heterotrimeric complex that possesses processive helicase and exonuclease activities. Upon encountering the DNA regulatory sequence, chi, the enzymatic properties of RecBCD enzyme are altered. Its helicase activity is reduced, the 3'-->5'nuclease activity is attenuated, the 5'-->3' nuclease activity is up-regulated, and it manifests an ability to load RecA protein onto single-stranded DNA. The net result of these changes is the production of a highly recombinogenic structure known as the presynaptic filament. Previously, we found that the recC1004 mutation alters chi-recognition so that this mutant enzyme recognizes an altered chi sequence, chi*, which comprises seven of the original nucleotides in chi, plus four novel nucleotides. Although some consequences of this mutant enzyme-mutant chi interaction could be detected in vivo and in vitro, stimulation of recombination in vivo could not. To resolve this seemingly contradictory observation, we examined the behavior of a RecA mutant, RecA(730), that displays enhanced biochemical activity in vitro and possesses suppressor function in vivo. We show that the recombination deficiency of the RecBC(1004)D-chi* interaction can be overcome by the enhanced ability of RecA(730) to assemble on single-stranded DNA in vitro and in vivo. These data are consistent with findings showing that the loading of RecA protein by RecBCD is necessary in vivo, and they show that RecA proteins with enhanced single-stranded DNA-binding capacity can partially bypass the need for RecBCD-mediated loading.

Figure 1. RecBCD and RecBC¹⁰⁰⁴D enzymes load RecA and RecA⁷³⁰ proteins onto χ*–containing ssDNA

A. Illustration of the substrate used and the major products produced by the helicase/nuclease activities of RecBCD enzyme upon χ recognition. B. Wild type RecBCD enzyme. Left: Gel showing RecA-loading onto χ-containing ssDNA, assayed by protection from exonuclease I digestion (present in all lanes except “-ExoI” controls). The proteins and linear dsDNA (χ⁺ or χ*) present are indicated at the top of the gel. The vertical arrows indicate the time of exonuclease I addition. Right: Quantification of χ–containing ssDNA protection. The amount of χ–containing ssDNA remaining is expressed relative to the initial amount of dsDNA (lane M): filled squares: RecA in the absence of exonuclease I (exp. set 1); filled circles: RecA (exp. set 2); open squares: RecA omitted (exp. set 3); and filled diamonds: RecA and χ^* instead of χ (exp. set 4). C. RecBC¹⁰⁰⁴D enzyme and χ^*. Left: Gel showing RecA-loading onto χ^*containing ssDNA, assayed by protection from exonuclease I digestion. The proteins (wild type RecA or RecA⁷³⁰) and linear dsDNA (χ*) present are indicated at the top of the gel. Right: Quantification of χ*–containing ssDNA protection: filled squares: RecA in the absence of exonuclease (exp. set 5); filled diamonds: RecA (exp. set 6); filled triangles: RecA⁷³⁰ (exp. set 7); and open squares: RecA omitted (exp. set 8). .

Figure 2. RecA⁷³⁰ possesses an enhanced capacity to form stable nucleoprotein filaments on SSB-ssDNA complexes

RecA nucleoprotein filaments were formed on heat-denatured linear dsDNA in the presence of SSB protein for comparison to nucleoprotein filament formation in the coupled RecA and RecBCD reactions. A. Wild type RecA protein and χ⁺ DNA. B. RecA⁷³⁰ protein and χ* DNA. The RecA proteins used in each experiment are indicated. The vertical arrows indicate the time of exonuclease I addition. Lanes marked “HD” and “M” represent heat-denatured and linear dsDNA, respectively. For comparison, experiments 1 and 4 are coupled reactions.

Figure 3. RecA⁷³⁰ protein can be loaded onto χ-containing ssDNA to form nucleoprotein filaments that are more stable than those formed by wild type RecA protein

A. Wild type RecBCD enzyme, RecA protein and χ⁺ DNA. “Coupled” experiments referred to reactions where RecA protein was present at the beginning of the DNA processing reaction by RecBCD enzyme. In “uncoupled” experiment, RecA protein was added after DNA processing by RecBCD enzyme. The vertical arrows indicate the time of exonuclease I addition. B. RecA⁷³⁰ protein, RecBC¹⁰⁰⁴D enzyme, and χ* DNA. C. Comparison of RecA⁷³⁰ with wild type RecBCD and χ versus wild type RecA with RecBC¹⁰⁰⁴D and χ*; both coupled and uncoupled reactions are shown.

Figure 4. Suppression of UV sensitivity by recA730

A. recA1 background. B. recF143 background. C. recF143 recC1004 background. D. recC1004 background. Open triangles represent the strains lacking any recA-expressing plasmid. Open squares represent the strains expressing wild type recA. Closed circles represent the strains expressing recA730; closed squares represent the wild type rec⁺ strains; closed diamonds represent the recC73 strains; and the closed triangles in panel C are recF143.

Figure 5. recA730 restores the recombination hotspot activity of χ* in a recF recC1004 strain

A. Design of the λ recombination crosses . Parental phages are defective for their phage-encoded recombination functions (red⁻ γ⁻⁻ int⁻). The S⁺ Jh recombinant phages were scored as to whether the plaque was turbid (cI⁺; crossover before the immunity region) or clear (cI⁻; crossover beyond the region). B. Recombination frequency. The relative value, normalized to the wild type (χ⁰) strain, of the ratio [S⁺ Jh recombinants/total phage] is plotted. Left panel is the recF143 recC1004 strain without plasmid (N = 3). Center panel is the same strain with the wild type recA expressing plasmid (N = 3). Right panel is the same strain with the recA730 expressing plasmid (N = 4). The recombination frequency and standard deviation for χ⁰ was 0.43 ± 0.08, 0.41± 0.14, and 0.43 ± 0.11 for the parental strain, wild type recA, and recA730, respectively. C. Hotspot activity. The ratio of turbid plaques to clear plaques is plotted.