(A) (left) Schematic of a representative casposon from Methanosarcina mazei (the gene organization of casposon MetMaz1FA1A3-C2 is shown; Krupovic et al., 2016) adjacent to host tRNA-Leu gene (red arrow). The last 4 bp of the tRNA-Leu gene overlap with 4 bp of the 14 bp target site duplication (TSD; green arrows) that flanks the casposon. The casposon Terminal Inverted Repeats (TIR) are shown as orange arrows. M. mazei casposon genes are as annotated by Krupovic et al. (2016), and are in gray with the exception of the putative Cas1-type casposase (in blue). Genes corresponding to a B-family type polymerase (‘polB’) and an HNH nuclease (‘HNH’) are marked. In the sequence below, a conserved 5'-CGCA motif at the 5'-end of the TSD is underlined. The sequence of the 31 bp TIR from strain S-6 and others, located at each casposon end, is shown in orange. (right) Relative to CRISPR Cas1, family two casposases have an additional C-terminal 60–70 residues predicted to form a helix-turn-helix (HTH) domain (Krupovic et al., 2014). Amino acid numbering is that of the M. mazei casposase. (B) In vitro integration assay of the M. mazei TIR into a target plasmid. The schematic at left shows the formation of relaxed and linear products corresponding to single-end (SE) and double-end (DE) integration of an oligonucleotide into a plasmid. The oligonucleotide is shown schematically in orange where the red dot indicates the 3'-OH nucleophile. In the agarose gels shown at the right, for the ‘no end’ controls, the plasmid was incubated for 60 min in the presence of protein and metal ion, but without the addition of the 31-mer TIR. For each reaction, the time points are: 0, 1, 5, and 60 min. Target: pUC19 (lanes 1–5); +14 bp TSD sequence (lanes 6–10); +TSD+15 bp more of the tRNA-Leu gene (lanes 11–15); +TSD+30 bp more of the tRNA-Leu gene (lanes 16–20).