protein, which is homologous to the C-terminal region of the human ERCC1 protein, is involved in DNA binding and 5′-incision

The incisions in the DNA at the 3′- and 5′-side of a DNA damage during nucleotide excision repair in Escherichia coli occur in a complex consisting of damaged DNA, UvrB and UvrC. The exact requirements for the two incision events, however, are different. It has previously been shown that the 3′-incision requires the interaction between the C-terminal domain of UvrB and a homologous region in UvrC. This interaction, however, is dispensable for the 5′-incision. Here we show that the C-terminal domain of the UvrC protein is essential for the 5′-incision, whereas this domain can be deleted without affecting the 3′-incision. The C-terminal domain of UvrC is homologous with the C-terminal part of the ERCC1 protein which, in a complex with XPF, is responsible for the 5′-incision reaction in human nucleotide excision repair. Both in the UvrC and the ERCC1 domain a Helix–hairpin–Helix (HhH) motif can be indicated, albeit at different positions. Such a motif also has been found in a large variety of DNA binding proteins and it has been suggested to form a structure involved in non-sequence-specific DNA binding. In contrast to the full length UvrC protein, a truncated UvrC protein (UvrC554) lacking the entire ERCC1 homology including the HhH motif no longer binds to ssDNA. Analysis of protein–DNA complexes using bandshift experiments showed that this putative DNA binding domain of UvrC is required for stabilisation of the UvrBC–DNA complex after the 3′-incision has taken place. We propose that after the initial 3′-incision the HhH motif recognises a specific DNA structure, thereby positioning the catalytic site for the subsequent 5′-incision reaction