An NMR study on the interaction of Escherichia coli DinI with RecA-ssDNA complexes

Abstract

The SOS response, a set of cellular phenomena exhibited by eubacteria, is initiated by various causes that include DNA damage-induced replication arrest, and is positively regulated by the co- protease activity of RecA. Escherichia coli DinI, a LexA-regulated SOS gene product, shuts off the initiation of the SOS response when overexpressed in vivo. Biochemical and genetic studies indicated that DinI physically interacts with RecA to inhibit its co-protease activity. Using nuclear magnetic resonance (NMR) spectroscopy, we show that DinI tightly binds to the central region of RecA (between the N- and C-terminal domains) and that this interaction is enhanced upon the oligomerisation of RecA. On the other hand, DinI did not inhibit the interaction between 4mer single-stranded (ss)DNA and RecA- ATPgammaS, but had a slight effect on the structure of ssDNA-RecA-ATPgammaS complexes involving 8mer and 12mer ssDNA. We hypothesise that prevention of repressor binding to the intermolecular cleft region of RecA protomers by DinI, with the possibility of a slight conformational change induced in the DinI-bound ssDNA-RecA-ATPgammaS complex, together function to inhibit the co-protease activity of RecA.

Figure 1

Solution structure of DinI. (A) The backbone traces of an ensemble of 20 refined structures superimposed on the backbone C_α, N and C′ atoms. (B) Ribbon representation of the structure closest to that of the mean of the ensemble. Molecular graphics images were produced using the MidasPlus software from the Computer Graphics Laboratory (University of California, San Francisco).

Figure 2

Physical interaction between fragments of RecA and DinI. Contour plots from ¹H-¹⁵N HSQC spectra of ¹⁵N-labelled DinI (A) in the absence of RecA, (B) in the presence of 0.1 mM RecA and (C) in the presence of 0.2 mM RecA_Δ1–33, showing the line broadening of DinI signals in the presence of RecA. All spectra were measured on 0.1 mM ¹⁵N-labelled DinI in 20 mM sodium phosphate, pH 6.5, containing 50 mM NaCl and 0.01% NaN₃ (90% H₂O:10% ²H₂O) at 20°C.

Figure 3

Differential effect of RecA fragments on signal intensities of ¹⁵N-labelled DinI. Changes in ¹H-¹⁵N cross-peak intensity for observable residues of [¹⁵N]DinI upon titration with increasing concentrations of (A) RecA_Δ1–33, (B) RecA_1–33 or (C) RecA_268–330 are plotted against the residue number. The intensity of each cross-peak was normalized relative to the intensity of the C-terminal residue 81. Data for overlapping cross-peaks and proline residues were excluded from these plots.

Figure 4

Interaction of DinI with 4mer ssDNA–RecA–ATPγS complex. Contour plots of NOE patterns of ssDNA from 2D transferred NOE spectra on the following samples: (A) ssDNA, (B) ssDNA with RecA, (C) ssDNA with DinI and (D) ssDNA with RecA and DinI are shown. The experiments were performed as described in Materials and Methods. The assignment of TRNOE cross-peaks was done according to Nishinaka et al. (24).

Figure 5

Influence of ssDNA length on the binding of DinI to ssDNA–RecA–ATPγS complex. 1D ¹H spectra of ssDNA (4mer, 8mer and 12mer) (A) in the absence of RecA, (B) in the presence of RecA and (C) in the presence of both RecA and DinI, indicating the narrowing of signals from DNA bases (ATPγS signals are indicated by arrows). Signals from RecA or DinI are too broad to be observed under this condition.

Figure 6

DinI influences the integrity of ssDNA–RecA–ATPγS complex. Contour plots of a similar region as in Figure 4, from TRNOE spectra of (A) ssDNA alone, (B) ssDNA in the presence of RecA and (C) ssDNA in the presence of both RecA and DinI. The changes with 8mer and 12mer ssDNA are represented on the left and right columns, respectively.