Characterization of the SMC-like plasmid defense system MksBEFG (a Wadjet type I system) of Corynebacterium glutamicum
For prokaryotes, horizontal gene transfer (HGT) is the main source of genetic exchange and recombination. Therefore, prokaryotic cells are constantly exposed to the risk of taking up foreign DNA. The most studied defense mechanisms are anti-phage systems. Recently, the focus shifted towards identifying unknown (anti-plasmid) systems. Especially, systems related to SMC-like complexes such as the MksBEFG (Wadjet) systems gained tremendous attention. Here, we investigated its molecular mechanism.
MksG is a novel, divalent cation-dependent nuclease effectively cleaving DNA in vitro in an ATP-independent manner. Structural analysis revealed a dimeric assembly through its C-terminal domain that is homologous to the TOPRIM domain of the Topo II family of enzymes. MksG contains the corresponding catalytic ion binding site essential for DNA cleavage by topoisomerase VI. Our data showed that MksG activity is not dependent on MksBEF in vitro, but the ATPase activity of the MksBEF complex is reduced in presence of MksG and plasmid DNA. Hence, we reason that this reaction cycle, in combination with the nuclease activity, allows for loop extrusion, subsequent activation and cleavage of the invading plasmid DNA.
Using SMLM & SPT, we determined the dynamics of the main enzymes. MksB is mainly confined at the cell poles. In contrast, MksG is present in three distinct populations: an ‘immobile’ population that comprises both DNA-bound and polarly anchored MksG, and two dynamic populations across the nucleoid and the cytosol. The presence of plasmid DNA directly changes MksG molecular dynamics. The polar localization of MksG is altered by MksBEF, which in turn colocalizes with DivIVA. Our data reveal that the plasmid defense mechanism is executed through the nuclease activity of MksG. It also provides evidence for the evolutionary connection between the interactions of Smc-like proteins and topoisomerases.
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