Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein.

How various macromolecules come together to form specific biological complexes and how the assembly/disassembly of such complexes are regulated are key to understanding any biological process. Characterization of such dynamical processes is challenging as it necessitates means to rapidly and specifically spark the assembly/disassembly aside from ways to examine the subsequent transformations over time. Recently, various chemical strategies have been developed to use light to trigger changes in oligonucleotide structures (and thus their activities). This study utilizes photoconvertible nucleic acid to trigger the biological assembly/disassembly of DNA damage repair proteins on DNA. Specifically, this work focuses on photo-regulation of DNA damage recognition by the xeroderma pigmentosum C complex (XPC-Rad23B; Rad4-23 in yeast). Rad4/XPC specifically recognizes various bulky helixdestabilizing/distorting lesions in the genomic DNA to initiate the global genome nucleotide excision repair (GG-NER) pathway in eukaryotes. This dissertation reports that 6-nitropiperonyloxymethyl (NPOM)-modified DNA can be used to modulate the DNA binding of the Rad4/XPC DNA repair complex using light. This collection of work exhibits NPOM recognized by the Rad4 protein as a specific substrate and such specificity can be abolished by light-induced cleavage of the NPOM group from DNA in a dose-dependent manner. In addition, NPOM-DNA conformational landscape is monitored by fluorescence lifetime technique and shows B-DNA-like conformations for NPOM adduct (despite its bulky modification) as verified by molecular dynamic studies. Further conformational analysis shows that Rad4-binding renders the NPOM-DNA conformation to be heterogeneously distorted. Furthermore, this work investigates the crystal structure of Rad4/XPC protein-bound NPOM-modified DNA and combination with fluorescence lifetime experiments reveals a novel mode of binding in which, Rad4 fails to open the DNA, instead is bound to the ends of DNA as opposed to previously seen ‘open conformation’ structure. This work also reports preliminary findings related to Rad4/XPC binding to two different types of DNA lesions, arylamine derivatives and, non-canonical DNA triplexes. Taken together, these studies suggest that photoactivable DNA may be used as a DNA lesion surrogate to study DNA repair mechanisms such as nucleotide excision repair and may help to provide mechanistic insights in Rad4/XPC lesion recognition of ‘repair-resistant’ DNA lesions.