Characterisation of the DNA-binding profile of barley HvCBF1 using an enzymatic method for rapid, quantitative and high-throughput analysis of the DNA-binding activity

A rapid and quantitative DNA-binding assay was developed based on the translational fusion of a DNA-binding protein (DBP) with a Neocallimastix patriciarum β-1,4-d-glucanase, CelD. CelD releases a fluorescent 4-methylumbelliferyl product from 4-methylumbelliferyl cellobioside. This hydrolysis activity was used to quantify the amount of DBP–CelD bound to an immobilised biotin-labelled target sequence. The DNA-binding assay can be performed in a 96-well plate format for high- throughput analysis of putative DBPs. This method was applied to analysis of the binding properties and sequence selectivity of a cold-inducible transcription factor HvCBF1 from barley containing an AP2 DNA-binding domain. A base-scanning approach using degenerate oligonucleotide probes was employed for rapid identification of the conserved core motif of the HvCBF1 binding site. Quantitative analysis of the binding site of HvCBF1 using systematic base substitution revealed that a (G/a) (C/t)CGAC sequence was sufficient to constitute a functional motif, where the lower-case letters represent less efficient bases. The method enables us to provide accurate and quantitative data on a comprehensive DNA-binding profile for a cold-inducible AP2 transcription factor as well as information on environmental parameters potentially influencing the DNA-binding activity. The accurate binding sequence data facilitate identification of candidate genes regulated by HvCBF1 from genome sequence databases.