Worm Breeder's Gazette 13(2): 103 (February 1, 1994)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

DNA binding properties of the Tc3 transposase protein.

Sean D. Colloms, Henri G.A.M. van Luenen, Ronald H.A. Plasterk

Figure 1

Figure 2

The Netherlands Cancer Institute, Division of Molecular Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

The transposable element Tc3 of Caenorhabditis elegans contains a single gene, Tc3A ,encoding a protein of 329 amino acids with 35% sequence identity to the transposase of Tc1 .Expression of Tc3A in a transgenic line leads to excision and transposition of Tc3 elements (Van Luenen et al. 1993. EMBO J. 12: 2513-2520).

Tc3A was expressed in Escherichia coli and found, by South-Western analysis using radio-labeled pUC18 DNA, to have a non-specific DNA binding activity. To ascertain which region of Tc3A is responsible for this activity, a series of carboxy-terminal deletion derivatives of Tc3A were expressed in E. coli. Tc3A 1-192 retained full DNA binding activity, whereas Tc3A 1-159 had only partial activity and Tc3A 1-98 showed no affinity for pUC18 DNA in a South-Western assay.

Tc3A carboxy-terminal deletion derivatives expressed in E. coli were found to have a specific DNA binding activity. Tc3A 1-98, Tc3A 1-85 and Tc3A 1-65 (but not Tc3A 1-54) bound to a DNA fragment containing the terminal 38 nucleotides of Tc3 ,giving retarded bands in a "band-shift" assay. In the same assay, Tc3A N-terminal derivatives did not bind to a DNA fragment containing Tc1 terminal sequences.

These results are consistent with those found for Tc1 transposase, which contains a non-specific DNA binding domain between amino acids 71 and 207, and a domain which binds specifically to Tc1 ends within the amino-terminal 63 amino acids (Vos et al. 1993. Genes and Dev 7: 1244-1253).

DNAse I footprinting experiments were used to determine the regions within the Tc3 inverted repeats to which the amino-terminal derivatives of Tc3A bind. Two regions of protection were found: between base-pairs 9 and 32 and also between base-pairs 182 and 205 relative to the transposon end. These two regions are within a 27 out of 31 bp match between the sequences 1-31 and 176-204 relative to the transposon end. Interactions between Tc3A amino-terminal derivatives were further analyzed by methylation interference experiments. Methylation at G and A residues in adjacent major and minor grooves (on one face of the helix) interfered with binding by Tc3A 1-65 and Tc3A 1-85. The DNAse I (brackets) and methylation interference (dots) results are summarized in figure B below, as are the positions at which Tc3A cuts at the transposon end (arrows; see WBG 13:1 p29 ).

The extent of DNA from the terminal inverted repeats required for transpostion was also investigated. A mini Tc3 element consisting of 80 bp from each end of Tc3 ,flanking a kanamycin resistance gene was capable of excision and transpostion when introduced into Bristol N2 together with an inducible copy of Tc3A .Thus, although two copies of a Tc3A binding site are present at each end of Tc3 ,the outermost Tc3A binding sites are sufficient for transposition of Tc3 .

Figure 1

Figure 2