Abstract
Mrr from Escherichia coli K12 is a type IV restriction endonuclease whose role is to recognize and cleave foreign methylated DNA. Beyond this protective role, Mrr can inflict chromosomal DNA damage that elicits the SOS response in the host cell upon heterologous expression of specific methyltransferases such as M.HhaII, or after exposure to high pressure (HP). Activation of Mrr in response to these perturbations involves an oligomeric switch that dissociates inactive homo-tetramers into active dimers. Here we used scanning number and brightness (sN&B) analysis to determine in vivo the stoichiometry of a constitutively active Mrr mutant predicted to be dimeric and examine other GFP-Mrr mutants compromised in their response to either M.HhaII activity or HP shock. We also observed in vitro the direct pressure-induced tetramer dissociation by HP fluorescence correlation spectroscopy of purified GFP-Mrr. To shed light on the linkages between subunit interactions and activity of Mrr and its variants, we built a structural model of the full-length tetramer bound to DNA. Similar to functionally related endonucleases, the conserved DNA cleavage domain would be sequestered by the DNA recognition domain in the Mrr inactive tetramer, dissociating into an enzymatically active dimer upon interaction with multiple DNA sites.
Highlights
Restriction endonucleases (REs) constitute a primary defense against invading phages or other foreign DNA entering bacterial cells [1,2]
We have proposed previously [19] that the active form of Mrr is a dimer, and that the activation mechanism upon high pressure (HP) shock that triggers the SOS-response is based on the pressure-induced dissociation of the inactive tetrameric form into the active dimer
Using scanning N&B in live E. coli cells, we compared the number and brightness of fluorescent particles detected in strains expressing wild-type green fluo rescent protein (GFP)-Mrr (WT Mrr) or the GFP-MrrN111S/D124G/V175G constitutive mutant (Mrr-Const), both of which were under the control of the arabinose-inducible PBAD promoter in a plasmid construct (Table S1)
Summary
Restriction endonucleases (REs) constitute a primary defense against invading phages or other foreign DNA entering bacterial cells [1,2]. In Escherichia coli K-12, Mrr (methylated adenine recognition and restric tion) has received special interest because of its involvement in the restricted cloning efficiency of methylated DNA isolated from plants and mammals [3]. More recently modification-dependent REs have emerged as useful tools for epigenetic studies [1,4,5]. Mrr, together with both E. coli methylcytosine restriction systems McrA and McrBC, belongs to the type IV family of REs that target modified, typically methylated, DNA with a loose sequence specificity [6]. Evidence that Mrr restricts methylated DNA comes from early observations of the mrr-dependent genotoxicity of various adenine or cytosine methyltransferases (MTases) such as M
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