Distinguishing characteristics of hyperrecombinogenic RecA protein from Pseudomonas aeruginosa acting in Escherichia coli

Abstract

In Escherichia coli, a relatively low frequency of recombination exchanges (FRE) is predetermined by the activity of RecA protein, as modulated by a complex regulatory program involving both autoregulation and other factors. The RecA protein of Pseudomonas aeruginosa (RecA(Pa)) exhibits a more robust recombinase activity than its E. coli counterpart (RecA(Ec)). Low-level expression of RecA(Pa) in E. coli cells results in hyperrecombination (an increase of FRE) even in the presence of RecA(Ec). This genetic effect is supported by the biochemical finding that the RecA(Pa) protein is more efficient in filament formation than RecA K72R, a mutant protein with RecA(Ec)-like DNA-binding ability. Expression of RecA(Pa) also partially suppresses the effects of recF, recO, and recR mutations. In concordance with the latter, RecA(Pa) filaments initiate recombination equally from both the 5' and 3' ends. Besides, these filaments exhibit more resistance to disassembly from the 5' ends that makes the ends potentially appropriate for initiation of strand exchange. These comparative genetic and biochemical characteristics reveal that multiple levels are used by bacteria for a programmed regulation of their recombination activities.

FIG. 1.

Visualization and quantitation of the RecA_Ec and RecA_Pa proteins by immunoblotting with a mixture of polyclonal antibodies raised against RecA_Ec and RecA_Pa. For controls, lanes RecAPa and RecAEc contain equal amounts of pure RecA proteins. Other lanes show the data from a typical experiment comparing the RecA_Pa and RecA_Ec amounts in different strains when protein was extracted from the same number of cells (for details, see Materials and Methods).

FIG. 2.

ssDNA-dependent ATP hydrolysis of four presynaptic complexes formed by RecA_Pa, RecA_Ec, RecA_Pa plus RecA K72R, and RecA_Ec plus RecA K72R at increasing concentrations of circular ssDNA. SSB was used at a concentration providing one SSB monomer per 10 nucleotides of ssDNA. Each point on the curves represents an individual sample and shows a steady-state rate measured over a period of about 15 min.

FIG. 3.

Time course of initiation of D-loop formation between circular supercoiled dsDNA and linear ssDNA with a region of homology at either the 3′ or 5′ end in a comparison of the characteristics of RecA_Pa and RecA_Ec in promotion of the reaction. The DNA substrates and the reactions are illustrated by schemes presented at the top of the figure. M13mp8.1037(+) circular ssDNA was linearized with either EcoRI or PstI to place the 1,037-nucleotide insert (filled rectangle) at the 5′ or 3′ end, respectively. The supercoiled M13mp8 dsDNA has 7,229 bp of homology to the 3′ end of EcoRI-digested M13mp8.1037(+) linear ssDNA or to the 5′ end of PstI-digested M13mp8.1037(+) linear ssDNA. The agarose gel assay was used to visualize joint molecule formation as described in Materials and Methods. (A) Agarose gel presentation of joint molecule formation. Lanes a, b, and c contain markers (M) of dsDNA, ssDNA, and dsDNA plus ssDNA, respectively. The positions of joint molecules (jm), linear ssDNA (ss), and closed circular dsDNA (ds) are shown to the left of the gels. (B) Quantitation of the data presented in panel A by use of the Kodak DS1D image analysis software. (C) Values calculated from the data presented in panel B and two other independent experiments. The bands obtained in the D-loop reactions proceeding 4, 6, and 8 min after initiation were summarized because they looked similar.

FIG. 4.

Time-course decrease in fluorescence after the addition of excess poly(dT) to the RecA/ATP/ɛDNA complex in a comparison of disassembly of RecA_Pa and RecA_Ec complexes. The presynaptic complex RecA/ATP/ɛDNA was formed through the primary DNA binding site of RecA (excess RecA compared to ɛDNA). Experiments were performed as described in Materials and Methods. After RecA_Pa and RecA_Ec complexes were preformed, the increase of fluorescence was established. Excess poly(dT) was then added to initiate the quenching of fluorescent signal (time zero) as result of the poly(dT) challenge. The measurements were started 0.5 min later. The broken lines are the extrapolation of data to the moment of poly(dT) addition to the reaction mixture. (Inset) The same data but presented in semilogarithmic scale. F/F₀ is a portion of fluorescence F of the RecA_Ec or RecA_Pa presynaptic complexes still existing to the time indicated; F₀ is the fluorescence at time zero.