Fig 1.
Identification of six Type III restriction-modification (RM) systems that methylate the N4 atoms of deoxycytosine bases.
Four systems share a common architecture with previously-identified Type III systems comprised of Res and Mod subunits, whereas two additional systems appear to encode a third protein subunit located 5’ of their respective mod genes. Note that in the case of the BspHIV system there is approximately 140bp of non-coding sequence between the 3’-end of the helicase-like ATPase subunit and the 5’-end of the mod gene. SMRT-sequencing was employed to identify derived consensus methylation motifs for each of the six Type III RM systems. The tall blue bars indicate the Inter-Pulse Duration (IPD) values associated with positions of N4C methylation.
Table 1.
Characterized m4C Type III R-M systems.
Fig 2.
Alignment of the six characterized Type III Methyltransferases that methylate the N4 atom of cytosine.
Each of the six Mod proteins are beta-class methyltransferases and contain conserved sequence motifs corresponding to the methyltransferase catalytic motif IV (DPPY) and the AdoMet binding motif I (FGG) described by Malone et al., [24].
Fig 3.
Alignment of the six characterized Type III endonucleases.
The Res subunit of each system contains a conserved Walker A (GSGKT) and Walker B DEAD-BOX/DEXH motif typically associated with ATP-dependent DNA helicase proteins, and a canonical PD-(D/E)XK endonuclease catalytic motif located toward the carboxy terminus.
Table 2.
Modification detection by SMRT sequencing.
Table 3.
LC-MS analyses of m4C modification for Type III systems.
Fig 4.
Eco86II endonuclease activity.
Partially purified Eco86II enzyme was used to digest pUC19 plasmid substrate containing 3 sites: one in the forward direction and two in reverse orientation. After cutting with Eco86II, the reaction was divided and cut with six REases that cleave pUC19 once at known positions. Although the Eco86II digestion is only partial, fragments having lengths corresponding to the distance between the known REases and the Eco86II cleavage positions are observed, demonstrating specific Eco86II cleavage downstream from its GAGCC recognition motif. The original gel photograph is available as S1 Raw image.
Table 4.
Putative m4C Type III R-M systems.