Crystallization of and selenomethionine phasing strategy for a SETMAR-DNA complex

Abstract

Transposable elements have played a critical role in the creation of new genes in all higher eukaryotes, including humans. Although the chimeric fusion protein SETMAR is no longer active as a transposase, it contains both the DNA-binding domain (DBD) and catalytic domain of the Hsmar1 transposase. The amino-acid sequence of the DBD has been virtually unchanged in 50 million years and, as a consequence, SETMAR retains its sequence-specific binding to the ancestral Hsmar1 terminal inverted repeat (TIR) sequence. Thus, the DNA-binding activity of SETMAR is likely to have an important biological function. To determine the structural basis for the recognition of TIR DNA by SETMAR, the design of TIR-containing oligonucleotides and SETMAR DBD variants, crystallization of DBD-DNA complexes, phasing strategies and initial phasing experiments are reported here. An unexpected finding was that oligonucleotides containing two BrdUs in place of thymidines produced better quality crystals in complex with SETMAR than their natural counterparts.

Keywords: DNA-binding domain; Hsmar1; SETMAR; crystallization; terminal inverted repeat; transposable element.

Figure 1

Oligonucleotides used for crystallization trials. Hsmar1 TIR-based DNA sequences were used for crystallization. The consensus Hsmar1 TIR sequence is shown for the left TIR element of the predicted ancestral transposon (top). The italic TA is the characteristic flanking dinucleotide in the Tc1/mariner transposon superfamily. Oligonucleotides screened include the 19 bp mariner binding site (shaded in gray) for TIR and the variant TIR sequences. Boxes indicate overhanging nucleotides expected to facilitate packing of the DNA in the lattice. Underlined bases indicate variations from the TIR sequence, and thymidines shown in bold in TIR2 and variant-TIR1 were replaced with bromodeoxyuridines for Br SAD phasing experiments.

Figure 2

Crystal images of initial crystallization trials. (a) DBD 329–440 (wild type) with TIR2. (b) DBD 329–440 (wild type) with TIR2 with TCEP additive. (c) DBD 329–440 (C381S) with TIR2. (d) DBD 329–440 (C381S) with variant TIR1. (e) DBD 316–440 (C381S) with variant TIR1. (f) DBD 329–440 (C381R) with TIR2.

Figure 3

Preliminary low-resolution electron-density map of a SETMAR DBD–DNA complex. (a) Experimental electron-density map phased at 4.2 Å resolution. Phases were calculated from three Se positions from data combined in BLEND from two SeMet-derivative crystals. (b) An initial phosphate backbone model is shown with the experimental density map.