An Aromatic Sensor with Aversion to Damaged Strands Confers Versatility to DNA Repair

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Abstract
It was not known how xeroderma pigmentosum group C (XPC) protein, the primary initiator of global nucleotide excision repair, achieves its outstanding substrate versatility. Here, we analyzed the molecular pathology of a unique Trp690Ser substitution, which is the only reported missense mutation in xeroderma patients mapping to the evolutionary conserved region of XPC protein. The function of this critical residue and neighboring conserved aromatics was tested by site-directed mutagenesis followed by screening for excision activity and DNA binding. This comparison demonstrated that Trp690 and Phe733 drive the preferential recruitment of XPC protein to repair substrates by mediating an exquisite affinity for single-stranded sites. Such a dual deployment of aromatic side chains is the distinctive feature of functional oligonucleotide/oligosaccharide-binding folds and, indeed, sequence homologies with replication protein A and breast cancer susceptibility 2 protein indicate that XPC displays a monomeric variant of this recurrent interaction motif. An aversion to associate with damaged oligonucleotides implies that XPC protein avoids direct contacts with base adducts. These results reveal for the first time, to our knowledge, an entirely inverted mechanism of substrate recognition that relies on the detection of single-stranded configurations in the undamaged complementary sequence of the double helix. DNA is constantly exposed to damaging agents such as ultraviolet light, carcinogens, or reactive metabolic byproducts causing thousands of DNA lesions in a typical human cell every hour. To prevent irreversible mutations, many of these different lesions are eliminated by a DNA repair system known as “nucleotide excision repair.” Repair is initiated by the XPC protein, which recognizes damaged sites in the DNA double helix. Here, we describe how the XPC protein probes the way in which the two DNA strands are aligned, and how a recurrent protein motif, termed oligonucleotide/oligosaccharide-binding fold, is used to detect dynamic fluctuations of DNA in the lesion containing regions. We show that XPC interacts preferentially with the undamaged strand opposite the lesion sites and conclude that XPC protein adopts an entirely indirect recognition mechanism to be able to detect a nearly infinite spectrum of DNA lesions.