Nucleotide excision repair is an important and highly conserved DNA repair mechanism with an exceptionally large range of chemically and structurally unrelated targets. Lesion verification is believed to be achieved by the helicases UvrB and XPD in the prokaryotic and eukaryotic processes, respectively. Using single molecule atomic force microscopy analyses, we demonstrate that UvrB and XPD are able to load onto DNA and pursue lesion verification in the absence of the initial lesion detection proteins. Interestingly, our studies show different lesion recognition strategies for the two functionally homologous helicases, as apparent from their distinct DNA strand preferences, which can be rationalized from the different structural features and interactions with other nucleotide excision repair protein factors of the two enzymes. Nucleotide excision repair (NER)4 is an important DNA repair mechanism with a large range of chemically and structurally unrelated targets. Examples range from bulky DNA adducts to interstrand DNA cross-links caused by antitumor drugs (1-3). In humans, NER is the only repair system for the removal of UV irradiation-induced photoproducts such as the intrastrand cross-linking cyclobutane-pyrimidine dimers (CPDs), and dysfunctional NER is responsible for severe diseases, including xeroderma pigmentosum (XP) (1-3).The mechanism of NER is highly conserved between organisms. In bacteria, NER involves the proteins UvrA, UvrB, and UvrC. In the current model of prokaryotic NER, a hetero-tetrameric complex of UvrA and UvrB (UvrA 2 B 2 ) scans the DNA for lesions. When a lesion is encountered, initial lesion sensing by a dimer of UvrA is based on detection of DNA distortion. Conformational changes in the UvrA 2 B 2 complex result in an unwinding and opening of dsDNA around the lesion, providing an unpaired (bubble) region likely required by UvrB to thread onto one of the ssDNA strands. The helicase UvrB is believed to verify the presence of a lesion by insertion of a ␤-hairpin via interactions with residues at the base of the hairpin (2). Upon lesion verification by UvrB, UvrA dissociates from the complex, and ATP re-binding by UvrB results in the formation of the lesion-specific UvrB-DNA complex, which recruits the NER endonuclease UvrC (UvrBC complex). UvrC carries out two incisions on either side of the lesion. The 12-13-nucleotide (nt)-long ssDNA stretch (2) containing the lesion can then be removed together with the endonuclease by the helicase UvrD, and the resulting gap is filled and sealed by DNA polymerase I and ligase.Eukaryotic NER encompasses a total of ϳ30 proteins, including the xeroderma pigmentosum group proteins (XPA-XPG). Repair can either be initiated by a stalled RNA polymerase in transcription-coupled NER or via global genome NER. In global genome NER, upon initial detection of short destabilized DNA structures by the CEN2-XPC-HR23B complex, the ATPase/ helicase XPB, which is part of the 10-subunit transcription factor IIH (TFIIH) complex, then directly interacts with XPC (4) and...

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different molecules and molecular assemblies in a heterogeneous sample. In the particular context of DNA interacting protein systems, different protein complex forms and their corresponding binding positions on target sites containing DNA fragments can thus be distinguished. Here, an application of AFM to the study of DNA lesion recognition in the prokaryotic and eukaryotic nucleotide excision DNA repair (NER) systems is presented. The procedures of DNA and protein sample preparations are described and experimental as well as analytical details of the experiments are provided. The data allow important conclusions on the strategies by which target site verification may be achieved by the NER proteins. Interestingly, they indicate different approaches of lesion recognition and identification for the eukaryotic NER system, depending on the type of lesion. Furthermore, distinct structural properties of the two different helicases involved in prokaryotic and eukaryotic NER result in and explain the different strategies observed for these two systems. Importantly, these experimental and analytical approaches can be applied not only to the study of DNA repair but also very similarly to other DNA interacting protein systems such as those involved in replication or transcription processes.

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