Journal
BIOIMPACTS
Volume 8, Issue 3, Pages 167-176Publisher
TABRIZ UNIV MEDICAL SCIENCES & HEALTH SERVICES
DOI: 10.15171/bi.2018.19
Keywords
DOF zinc finger domain; DNA binding affinity; Gel retardation assay; Microscale thermophoresis
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Funding
- Research Office of Tabriz University of Medical Sciences [92/4-5/2]
- School of Life and Environmental Sciences, The University of Sydney
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Introduction: DOF proteins are a family of plant-specific transcription factors with a conserved zinc finger (ZF) DNA-binding domain. Although several studies have demonstrated their specific DNA binding, quantitative affinity data is not available for the binding of DOF domains to their binding sites. Methods: ZF domains of DOF2.1, DOF3.4, and DOF5.8 from Arabidopsis thaliana were expressed and purified. Their DNA binding affinities were assessed using gel retardation assays and microscale thermophoresis with two different oligonucleotide probes containing one and two copies of recognition sequence AAAG. Results: DOF zinc finger domains (DOF-ZFs) were shown to form independently folded structures. Assessments using microscale thermophoresis demonstrated that DOF-ZFs interact more tightly (similar to 100 fold) with double-motif probe than the single-motif probe. The overall K-d values for the DOF3.4-ZF and DOF5.8-ZF to the double-motif probe were similar to 2.3 +/- 1 and 2.5 +/- 1 mu M, respectively. Conclusion: Studied DOF-ZF domains formed stable complexes with the double-motif probe. Although DOF3.4-ZF and DOF5.8-ZF do not dimerize with an appreciable affinity in the absence of DNA (judging from size-exclusion and multiangle laser light scattering data), it is possible that these ZFs form protein-protein contacts when bound to this oligonucleotide, consistent with previous reports that DOF proteins can homo-and hetero-dimerize.
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