New in press
Cells are continuously facing the risk of taking up foreign DNA that can compromise genomic integrity. Therefore, bacteria are in a constant arms race with mobile genetic elements such as phages, transposons and plasmids. They have developed several active strategies against invading DNA molecules that can be seen as a bacterial ‘innate immune system’. Here, we investigated the molecular arrangement of the Corynebacterium glutamicum MksBEFG complex, which is homologous to the MukBEF condensin system. We show here that MksG is a nuclease that degrades plasmid DNA. The crystal structure of MksG revealed a dimeric assembly through its C-terminal domain that is homologous to the TOPRIM domain of the topoisomerase II family of enzymes and contains the corresponding ion binding site essential for DNA cleavage in topoisomerases. The MksBEF subunits exhibit an ATPase cycle in vitro and we reason that this reaction cycle, in combination with the nuclease activity provided by MksG, allows for processive degradation of invading plasmids. Super-resolution localization microscopy revealed that the Mks system is spatially regulated via the polar scaffold protein DivIVA. Introduction of plasmids results in an increase in DNA bound MksG, indicating an activation of the system in vivo.
MksG is the active nuclease subunit of the defense system.
Nuclease activity assays of MksG dimer, 5 μM protein were incubated 1 h at 30°C with 250 ng plasmid DNA (pJC1, 6108 bp) and 10 mM different divalent Metal-ions (second lane with Mn2+ only 1 mM). Reaction was stopped by adding 6x purple loading dye (NEB) and boiling samples 5 min at 90°C. DNA was separated on an agarose gel in TAE buffer. Assays for mutants (MksG (E236A), MksG (D279A), MksG (Y258A)) were incubated 3 h at 30°C.
Published in Nucleic Acids Research; https://doi.org/10.1093/nar/gkad130
Weiß M, Giacomelli G, Assaya MB, Grundt F, Haouz A, Peng F, Petrella S, Wehenkel AM, Bramkamp M
Nucleic Acids Res. 2023 Mar 7:gkad130. doi: 10.1093/nar/gkad130
Guo L, Sattler, L, Shafqat S, Graumann PL, Bramkamp M
mBio, 2022 13 (1):e0375321. doi: 10.1128/mbio.03753-21
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