Uracil-DNA glycosylase as a probe for protein-DNA interactions

Abstract

The DNA repair enzyme Uracil-DNA Glycosylase (UDG) can be used to investigate three different features of protein-DNA interactions. Complexes can be probed by simple protection experiments ('footprinting') or by two kinds of interference assays: a missing thymine site (MT-site) experiment and a missing thymine methyl site (MTM-site) experiment. The three probing methods are assessed using the well-characterized in vitro systems of lambda repressor and lac repressor binding to their respective operator sites. The results obtained with UDG probing agree well with previous probing experiments on the same systems and, in certain cases, extend previous interpretations: for example, comparison of the results obtained with the two interference assays shows that formation of the lac repressor-operator complex requires interactions with the methyl group of one particular thymine residue (T-13) in the operator but also requires interactions with other parts of the thymine base at operator positions 7, 8, 9, 21, 23 and 24. Overall, the properties of UDG recommend it as a versatile and convenient method to investigate DNA-protein interactions both in vitro and possibly in vivo.