Research Article

Poor Homologous Synapsis 1 Interacts with Chromatin but Does Not Colocalise with ASYnapsis 1 during Early Meiosis in Bread Wheat

Figure 3

TaPHS1 interacts with DNA in vitro. An E. coli BL21 cell line containing the pDEST17-TaPHS1 ORF construct was induced with IPTG (1 mM total concentration) to heterologously produce His-tagged TaPHS1 protein. Total cellular protein was extracted under native conditions, and the His-tagged TaPHS1 was isolated and purified using nickel affinity chromatography. Total cellular protein from the same cell line which was not induced was also extracted and treated identically to be used as the negative control. DNA-binding ability was only observed in assays conducted using the full-length TaPHS1 and the Region 1 peptide, indicating that Region 1 possesses a novel/uncharacterised DNA-binding domain where two S/TPXX putative DNA-binding motifs are located. Using competitive DNA-binding assays with equivalent amounts of single- and double-stranded DNA, TaPHS1 preferentially binds single-stranded DNA (ssDNA). This is evidenced by the increased retardation of the ssDNA species through the gel matrix with increasing concentrations of TaPHS1 that caused more ssDNA to be bounded by TaPHS1. At higher concentrations, TaPHS1 also interacts with double-stranded DNA (dsDNA). (a) Full-length TaPHS1, (b) Region 1 peptide, (c) Region 2 peptide, (d) Region 3 peptide, and (e) Region 4 peptide. Competitive DNA-binding assays performed with the induced samples containing the purified TaPHS1 protein are on the left and noninduced controls are on the right. μM: protein concentration, L: Ladder.