High Pressure Induced DNA Damage in Escherichia Coli Involves Pressure-Mediated Dissociation of the Tetrameric Mrr Restriction Endonuclease
Pressure shock of ∼100 MPa elicits a bona fide DNA damage response in the mesophilic E. coli bacterium, despite the fact that pressure, itself, cannot compromise DNA integrity. Screens for resistance to pressure shock by Aertsen's group revealed an endogenous restriction endonuclease, Mrr (Methylated adenine Recognition and Restriction), that cleaves methylated DNA. Since expression of Mrr is not toxic for the cell in the absence of HP, pressure stress must somehow be able to elicit Mrr activity. We used fluorescence fluctuation microscopy of strains chromosomally expressing a GFPmut2-Mrr fusion to investigate HP activation of Mrr. In scanning mode, FFM provides a spatial map of the absolute concentration (=n/PSF) and stoichiometry (=brightness, e) of the fluorescent particles. Before HP shock GFPmut2-Mrr exhibits a stoichiometry consistent with a tetramer. It appears to be slightly preferentially localized around the chromosome, diffusing more slowly than free GFP, yet not being immobile. After 20 min at 100 MPa followed by pressure release, GFPmut2-Mrr is found accumulated in foci that co-localize strongly with the chromosome, as previously observed. Interestingly, the mobility of the Mrr in these foci is significantly decreased and their stoichiometry is consistent with a dimer. The Mrr molecules which are excluded from the foci remain tetrameric. We suggest that pressure-mediated triggering of Mrr activity and the concomitant generation of DNA damage involves the conversion of inactive Mrr tetramers to active Mrr dimers. This could occur either via a direct effect of pressure on the Mrr tetramer-dimer equilibrium, a common effect of pressure on oligomers, or via a pressure effect on chromosome structural that leads to more efficient recruitment of active dimeric Mrr.