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. 2006 May 3;103(21):8101–8106. doi: 10.1073/pnas.0601161103

Fig. 3.

Fig. 3.

In vitro DNA-binding activity and specificity of the MAR domain of SETMAR. (a) Schematic representation of the SETMAR protein and its predicted features: pre-SET (p-S), helix–turn–helix motif (HTH), and DDN triad (positions of the original catalytic amino acid triad of the MAR region). The protein multiple alignment on the right shows that the triad is DD34N (∗) in all of the SETMAR protein sequences examined (naming convention as in Fig. 2) instead of the typical DD34D motif of the Hsmar1 and Hsmar2 consensus transposase sequences (14, 22) and all known active mariner transposases, such as mos1 from Drosophila melanogaster (Mos1-Dm). Dots indicate identity with top sequence, and numbers indicate the number of amino acids between the sequence portions shown. (b) In vitro DNA-binding activity and specificity of purified MAR protein domain. EMSA of various TIR double-stranded oligonucleotides mixed with a purified recombinant peptide corresponding to MBP domain alone (top lane) or to the entire MAR region fused to a N-terminal MBP domain (all other lanes). The TIR oligonucleotides were designed by using the consensus Hsmar1 or Hsmar2 sequences (14, 22) and their characteristic flanking TA target site duplication. Base substitutions relative to the Hsmar1 TIR are in bold and underlined. The EMSA autoradiography shows shifted DNA (bound) on the right side of the gel, whereas input DNA (unbound) is on the left side. MARx7/8 corresponds to a mixture of two oligonucleotides, none of which are bound by the purified protein. (c) Mapping of the MAR region involved in DNA binding. EMSA of either the Hsmar1 or Hsmar2 TIR oligonucleotides with four recombinant purified peptides corresponding to the entire MAR peptide (lane 1), the first 126 (lane 2) or 92 (lane 3) aa of the MAR peptide fused to a N-terminal MBP tag, or the MBP alone (lane 4). Two shifted bands can be seen when the Hsmar1 TIR oligonucleotide is mixed with either peptide 1 or peptide 2. Based on previous in vitro studies of mariner DNA-binding activities (23–26), we interpret complex (Cplx) 3 as a single oligonucleotide with a protein dimer, whereas the upper bands may correspond to tetramers of protein bound to single (Cplx 2) or paired (Cplx 1) oligonucleotides.