FLOW LINEAR DICHROISM AND ELECTRON-MICROSCOPIC ANALYSIS OF PROTEIN-DNA COMPLEXES OF A MUTANT UVRB PROTEIN THAT BINDS TO BUT CANNOT KINK DNA

(A)BC excinuclease of Escherichia coli is the enzymatic activity resul ting from sequential and partially overlapping actions of UvrA, UvrB, and UvrC protein. UvrA is a molecular matchmaker which promotes the fo rmation of a stable UvrB-damaged DNA complex in which the DNA is kinke d by about 130 degrees. The UvrB-DNA complex is then recognized by Uvr C) and two incisions are made in the DNA by the joint actions of UvrC and UvrB. A mutant of UvrB (D478A) can be loaded onto the DNA but it d oes not interact with UvrC to cause a nick 3' to the lesion. Based on the lack of a DNase-I-hypersensitive site in the footprint of the muta nt, it was proposed that the lack of incision was due to the inability of the mutant UvrB to kink the DNA. In the current study we have inve stigated the interaction of the mutant UvrB with DNA using two biophys ical methods, flow linear dichroism and electron microscopy. Both meth ods reveal that the mutant UvrB is unable to bend DNA.