ACCESSIBILITY OF EPITOPES ON UVRB PROTEIN IN INTERMEDIATES GENERATED DURING INCISION OF UV-IRRADIATED DNA BY THE ESCHERICHIA-COLI UVR(A)BC ENDONUCLEASE

Structural intermediates generated during incision of damaged DNA by t he Uvr(A)BC endonuclease were probed with monoclonal antibodies (mAbs) raised against the Escherichia coli UvrB protein. It was found that t he epitope of B2C5 mAb, mapped at amino acids (aa) 171-278 of UvrB, is not accessible in any of the preformed Uvr intermediates. Preformed B 2C5-UvrB immunocomplexes, however, inhibited formation of those interm ediates. B2C5 mAb seems to interfere with the formation of the UvrA-Uv rB complex due to overlapping of its epitope and the UvrA binding regi on of UvrB, Conversely, the epitope of B3C1 mAb (aa 1-7 and/or 62-170) was accessible in all Uvr intermediates. The epitope of B2E3 mAb (aa 171-278) was not accessible in any of the nucleoprotein intermediates preceding UvrB-DNA preincision complex. However, B2E3 was able to im munoprecipitate this complex and to inhibit overall incision. B2A1 mAb (aa 8-61) inhibited formation of those Uvr intermediates requiring AT P binding and/or hydrolysis by UvrB, B2B9 mAb (aa 473-630) inhibited Uvr nucleoprotein complexes involving UvrB, B2B9 seems to prevent the binding of the UvrA-UvrB complex to DNA, The epitope of the B3E11 mA b (aa 379-472) was not accessible in Uvr complexes formed at damaged s ites. These results are discussed in terms of structure-functional map ping of UvrB protein.