A model for the abrogation of the SOS response by an SOS protein: a negatively charged helix in DinI mimics DNA in its interaction with RecA

Abstract

DinI is a recently described negative regulator of the SOS response in Escherichia coli. Here we show that it physically interacts with RecA and prevents the binding of single-stranded DNA to RecA, which is required for the activation of the latter. DinI also displaces ssDNA from a stable RecA-DNA cofilament, thus eliminating the SOS signal. In addition, DinI inhibits RecA-mediated homologous DNA pairing, but has no effect on actively proceeding strand exchange. Biochemical data, together with the molecular structure, define the C-terminal alpha-helix in DinI as the active site of the protein. In an unusual example of molecular mimicry, a negatively charged surface on this alpha-helix, by imitating single-stranded DNA, interacts with the loop L2 homologous pairing region of RecA and interferes with the activation of RecA.

Figure 1

Physical interaction between the RecA and DinI proteins is detected by coimmunoprecipitation. In all cases either monoclonal anti-RecA ARM193 or polyclonal affinity-purified anti-DinI antibodies, in combination with the ELC detection system, were used for immunodetection of RecA and DinI proteins. (A) RecA–DinI complex was formed in the presence of ssDNA and ATPγS. Products of immunoprecipitation performed with either preimmune (lanes 2–4) or anti-DinI sera (lanes 5–7) were separated on two tricine SDS gels. As controls, RecA alone (lanes 2,5) and DinI alone (lanes 3,6) were subjected to the identical procedure. Lanes 1 and 8 contain purified RecA and DinI. RecA coimmunoprecipitated with DinI is visible in lane 7. (B) Immunoprecipitation of RecA–DinI complex formed in the presence of different combinations of ATPγS, ADP, and ssDNA was performed with antigen-purified anti-DinI antibody. Higher molecular weight bands on the RecA blot correspond to covalently crosslinked RecA dimers formed during prolonged incubation in the absence of reducing agent. The low intensity RecA signal visible in lane 2 is a result of RecA aggregation (see Materials and Methods).

Figure 2

The affinity of RecA and DinI for each other increases late in the SOS response. (A) Kinetics of expression of the DinI and RecA proteins in E. coli cell lysates prepared from UV-irradiated and untreated cultures. Wild-type K12 cells were allowed to recover for different periods of time after irradiation with 25 J/m² of 254 nm UV light, and cell lysates were prepared. Ten micrograms (for DinI) or 1 μg (for RecA) of total protein were used for Western blot analysis. Polyclonal affinity-purified anti-DinI antibody and anti-RecA serum were used for DinI and RecA detection, respectively. (B) Coimmunoprecipitation of DinI and RecA with anti-DinI antibody. Top panel shows original data obtained with the same lysates and antibodies used in A. RecA controls correspond to mock experiments in which anti-DinI antibody was omitted at the immunoprecitation step and anti-RecA serum was used for detection. Left panel represents relative amounts (in AU or arbitrary units) of DinI and RecA recovered at different time points from irradiated cells. Right panel shows recovery of RecA normalized to the amount of immunoprecipitated DinI. The normalized signal represents the ratio between the RecA and DinI signals in arbitrary units.

Figure 3

DinI specifically inhibits the ssDNA-binding activity of RecA. (A) Addition of DinI suppresses ssDNA-binding activity of RecA, but not SSB protein, as revealed by filter binding. 0.5 μM RecA or SSB was first preincubated with increasing concentrations of DinI for 10 min at room temperature. After addition of 0.5 μM ³²P-labeled 53-mer BS-S1 oligonucleotide, the binding reaction was allowed to proceed at 37°C for 30 min (the buffer conditions are described in Materials and Methods). (B) Displacement of intact ssDNA from ternary ssDNA–RecA–DinI complexes by DinI can be detected by electrophoresis on agarose gels. 2.5 μM ³²P-labeled BS-S1 was first preincubated with 0–60 μM DinI at 37°C for 30 min, then 1 μM RecA was added and the reaction was further incubated at 37°C for 30 min. Samples were separated on 1.2% agarose. (C) Time of DinI addition determines extent of inhibition of binding of RecA to ssDNA. The reactions contained the same amounts of ssDNA, RecA, and DinI as in B, but were assembled in three different orders of addition. (1) Prevention: RecA was incubated with DinI first, then ssDNA was added. (2) Interference: DinI and ssDNA were preincubated together and then allowed to compete for RecA. (3) Displacement: DinI was added to preformed stable ssDNA–RecA complexes. In each case, two constituents of the reaction were preincubated at 37°C for 30 min, and after addition of the third component, reactions were allowed to proceed for an additional 30 min. After separation on 1.2% agarose gels, the amount of free DNA was quantitated and plotted as a percentage of the total radioactivity in the lane.

Figure 4

The loop L2-derived peptide from RecA (WECO) interacts with the C-terminal peptide from DinI. DinI and all DinI peptides were present at a concentration of 25 μM in the DNA-binding reaction; the concentration of ssDNA was 0.5 μM. WECO is a loop L2 derived 20-mer peptide comprising the DNA-binding and pairing domain of RecA (Voloshin et al. 1996; Wang et al. 1998). (A) The whole DinI protein and the DinI 56–78 peptide derived from its C terminus prevent binding of WECO to ssDNA and disassemble the ssDNA–WECO complex. (B) Interaction between WECO and DinI 56–78 is specific because peptides with the same amino acid composition but scrambled sequence, SCR1 and SCR2, do not disrupt the WECO–ssDNA complex.

Figure 4

The loop L2-derived peptide from RecA (WECO) interacts with the C-terminal peptide from DinI. DinI and all DinI peptides were present at a concentration of 25 μM in the DNA-binding reaction; the concentration of ssDNA was 0.5 μM. WECO is a loop L2 derived 20-mer peptide comprising the DNA-binding and pairing domain of RecA (Voloshin et al. 1996; Wang et al. 1998). (A) The whole DinI protein and the DinI 56–78 peptide derived from its C terminus prevent binding of WECO to ssDNA and disassemble the ssDNA–WECO complex. (B) Interaction between WECO and DinI 56–78 is specific because peptides with the same amino acid composition but scrambled sequence, SCR1 and SCR2, do not disrupt the WECO–ssDNA complex.

Figure 5

Negatively charged surface on the C-terminal α-helix in DinI mimics DNA. Figure is prepared with RasMol program from the structure determined by Ramirez and coworkers (2000). (A) Overall three-dimensional structure of DinI. (B,C) Space-filling representation of the negatively charged residues in the active site of DinI. Image (C) is rotated by 90° with respect to projection (B) to emphasize that the highlighted acidic residues form a surface.

Figure 6

Electrostatic contacts between loop L2 of RecA and the C-terminal α-helix in DinI are critical to the interaction between RecA and DinI. The binding of wild-type and loop L2 mutants of RecA to wild-type DinI (A) and DinI_E72A (B) was monitored using surface plasmon resonance on a BIAcore 1000 instrument. The data are presented in the form of sensorgrams that represent the changes in the surface plasmon resonance signal as a function of time.

Figure 6

Electrostatic contacts between loop L2 of RecA and the C-terminal α-helix in DinI are critical to the interaction between RecA and DinI. The binding of wild-type and loop L2 mutants of RecA to wild-type DinI (A) and DinI_E72A (B) was monitored using surface plasmon resonance on a BIAcore 1000 instrument. The data are presented in the form of sensorgrams that represent the changes in the surface plasmon resonance signal as a function of time.

Figure 7

Mutations in the C-terminal α-helix of the DinI protein decrease the UV sensitivity of E. coli SY183 cells (Yasuda et al. 1998) overexpresing plasmid-born DinI. Cells expressing DinI mutant proteins with two acidic amino acids replaced with alanine (B) are less UV sensitive than cells expressing mutant proteins in which a single acidic amino acid has been replaced (A). SY183 carrying pBluescript II SK+ (Vector) or pBluescript II SK+ with the wild-type dinI gene (WT) were used as controls. Details of the experiment are described in Materials and Methods.

Figure 7

Mutations in the C-terminal α-helix of the DinI protein decrease the UV sensitivity of E. coli SY183 cells (Yasuda et al. 1998) overexpresing plasmid-born DinI. Cells expressing DinI mutant proteins with two acidic amino acids replaced with alanine (B) are less UV sensitive than cells expressing mutant proteins in which a single acidic amino acid has been replaced (A). SY183 carrying pBluescript II SK+ (Vector) or pBluescript II SK+ with the wild-type dinI gene (WT) were used as controls. Details of the experiment are described in Materials and Methods.

Figure 8

DinI inhibits RecA-mediated homologous DNA pairing but does not prevent completion of the actively proceeding strange exchange reaction. (A) Schematic illustration of the experiment. Presynaptic filament was formed after incubation of unlabeled BS-S1 with a stoichiometric amount of RecA followed by addition of DinI; the pairing reaction was immediately initiated by addition of homologous BS duplex. (B) The amount of displaced ³²P-labeled BS-S1 strand, reflecting the extent of the pairing reaction, was estimated after deproteinization and separation of the samples by 12% native PAGE. (C) Quantitation of the gel shown in B. (D) Illustration of the strand exchange reaction. RecA mediates pairing of covalently closed ssDNA (CSS) and homologous linear dsDNA (L) giving rise to joint molecules (JM). RecA-promoted branch migration results in strand exchange and formation of double-stranded open circles (OC) and linear ssDNA (LSS). Substrates and the final product of the reaction (OC) can be easily separated on an agarose gel. (E) Time course of the standard (DinI-free) strand exchange reaction. (F) Addition of DinI to the strand exchange reaction before or right after dsDNA blocks strand transfer. However, the yield of the final product (OC) was not affected when DinI was introduced to the reaction after DNA pairing had occurred.

Figure 9

The possible role of DinI in the SOS regulatory network. The SOS response is signaled on by RecA binding to ssDNA that accumulates in the cell as a result of DNA damage. Induction of the SOS response leads to overexpression of at least 31 gene products (Fernandez de Henestrosa et al. 2000). Among them are RecA, LexA, and DinI, the proteins involved in the autoregulation of SOS. The role of DinI consists in turning down the SOS response by abolishing the SOS signal. DinI can eliminate this signal in two ways: by preventing interaction between RecA and ssDNA or by disrupting ssDNA–RecA cofilaments. A detailed description of the SOS response and its induction can be found in several reviews (Friedberg et al. 1995; Walker 1996; Kuzminov 1999).