UV light-damaged DNA and its interaction with human replication protein A: an atomic force microscopy study

We have imaged a non-damaged and UV-damaged DNA fragment and its complexes with human replication protein A (RPA) using tapping mode atomic force microscopy (AFM). For imaging, molecules were immobilized under nearly physiological conditions on mica surfaces. Quantitative sizing of the 538 bp DNA before and after UV light treatment shows a reduction in the contour and persistence lengths and mean square end-to-end distance as a consequence of UV irradiation. Complexes of the UV-damaged DNA with RPA, an essential component of the initial steps of nucleotide excision repair, can be detected at high resolution with AFM and reveal conformational changes of the DNA related to complex formation. By phase image analysis we are able to discriminate between protein and DNA in the complexes. The DNA molecules are found to ‘wrap’ around the RPA, which in turn results in a considerable reduction in its apparent contour length.