Regulation of endonuclease activity by proteolysis prevents breakage of unmodified bacterial chromosomes by type I restriction enzymes

Abstract

ClpXP-dependent proteolysis has been implicated in the delayed detection of restriction activity after the acquisition of the genes (hsdR, hsdM, and hsdS) that specify EcoKI and EcoAI, representatives of two families of type I restriction and modification (R-M) systems. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However, unmodified targets may be generated in the DNA of an hsd(+) bacterium as the result of replication errors or recombination-dependent repair. We show that ClpXP-dependent regulation of the endonuclease activity enables bacteria that acquire unmodified chromosomal target sequences to survive. In such bacteria, HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP. A mutation that blocks only the modification activity of EcoKI, leaving the cell with approximately 600 unmodified targets, is not lethal provided that ClpXP is present. Our data support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences, but before completion of the pathway that would result in DNA breakage.

Figure 1

Restriction of unmodified phage λ by clp⁺ (NK301, NK300 for nalidixic acid) and clpX (NK304, NK320 for nalidixic acid) bacteria. Only clp⁺ cells show restriction alleviation.

Figure 2

Restriction of unmodified phage λ by dam (NK302), mutD (NK326), and topA (RS2) strains and their clpX derivatives (NK315, NK327, and NK329). It is known that topA strains accumulate compensatory mutations in gyrA or gyrB (21), but the topA10 strain (RS2) is not known to have a compensatory mutation (21), and the topA mutation itself correlates with impaired restriction (G. P. Davies, personal communication).

Figure 3

Assays for HsdR and HsdM polypeptides after treatment with 2-AP. (a) clp⁺ bacteria (NK301). (b) clpX bacteria (NK304). In the absence of 2-AP (data not shown), the assays for clp⁺ and clpX bacteria were indistinguishable from those seen in b. EcoKI polyclonal antibody, used in these Western blots, fails to detect HsdS, but detects HsdR and HsdM and some other E. coli proteins.

Figure 4

The stability of HsdR in vivo after treatment with 2-AP. Labeled polypeptides separated by electrophoresis through SDS-polyacrylamide gels (6%) were detected by autoradiography. An extract from a strain lacking HsdR (NK311/pACYC184) was analyzed in the first track. Samples from clp⁺ and clpX bacteria containing pNK3 were taken at the time intervals indicated after pulse labeling.

Figure 5

Hsd subunits were monitored, after treatment with 2-AP, using antibodies raised against the relevant R-M complex. HsdR is degraded only when it is a part of a functional complex. (a) Degradation of HsdR is prevented by a missense mutation in hsdR (track 5) or by the absence of HsdM and S (track 8). (b) The presence of functional EcoAI has no effect on the degradation of the HsdR subunit of EcoKI (Upper), even though the HsdR subunit of EcoAI itself is degraded (Lower, lanes 2, 5, and 8). The control tracks for EcoAI contain a mixture of polypeptides in which HsdM and HsdS are present in molar excess to give strong signals with antibody.

Figure 6

The survival of hsdR⁺M⁻(F269G)S⁺ cells was assessed after the conjugative transfer of hsdR⁺ to hsdR⁻M⁻S⁺ recipients. (a) The experiment using F′101–103 (hsdR⁺). (b) The control experiment with F′101–102 (hsdM⁺S⁺). Data are plotted for the following recipients: NK379, hsdR (▴); NK380, hsdR clpX (▿); NK382, hsdRM (□); NK384, hsdRM clpX (■); and NK383, hsdRM recA (●). The data show that hsdR⁺M⁻(F269G)S⁺ cells survive only if the recipient is ClpX⁺.

Figure 7

The effect of hsdM (F269G) on the level of HsdR. The mutation hsdM (F269G) destroys only the modification activity of EcoKI. The level of HsdR was monitored by Western blots by using antibody against EcoKI. Lanes 1–6 include extracts of strains. Lanes: 1, NK301 (hsd⁺); 2, NK386 [an hsdM (F269G) derivative of NK301]; 3, an hsd⁺ derivative of NK386; 4, an hsdR(A619V) derivative of NK386 (NK388) in which alleles of hsd genes were replaced by using λhsd phages that included only hsdMS or hsdR, respectively; 5, NM802 (an hsdR deletion strain); and 6, NK352 (an hsdMS deletion strain).