URACIL-DNA GLYCOSYLASE AS A PROBE FOR PROTEIN - DNA INTERACTIONS

The DNA repair enzyme Uracil-DNA Glycosylase (UDG) can be used to inve stigate three different features of protein - DNA interactions. Comple xes 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. T he three probing methods are assessed using the well-characterized in vitro systems of lambda repressor and lac repressor binding to their r espective operator sites. The results obtained with UDG probing agree well with previous probing experiments on the same systems and, in cer tain cases, extend previous interpretations: for example, comparison o f the results obtained with the two interference assays shows that for mation of the lac repressor - operator complex requires interactions w ith the methyl group of one particular thymine residue (T-13) in the o perator but also requires interactions with other parts of the thymine base at operator positions 7, 8, 9, 21, 23 and 24. Overall, the prope rties of UDG recommend it as a versatile and convenient method to inve stigate DNA - protein interactions both in vitro and possibly in vivo.