Conservation and Divergence in Nucleotide Excision Repair Lesion Recognition.

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.

Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging.

The ability of DNA glycosylases to rapidly and efficiently detect lesions among a vast excess of nondamaged DNA bases is vitally important in base excision repair (BER). Here, we use single molecule imaging by atomic force microscopy (AFM) supported by a 2-aminopurine fluorescence base flipping assay to study damage search by human thymine DNA glycosylase (hTDG), which initiates BER of mutagenic and cytotoxic G:T and G:U mispairs in DNA. Our data reveal an equilibrium between two conformational states of hTDG-DNA complexes, assigned as search complex (SC) and interrogation complex (IC), both at target lesions and undamaged DNA sites.

Strand-specific recognition of DNA damages by XPD provides insights into nucleotide excision repair substrate versatility.

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts.

DNA substrate preparation for atomic force microscopy studies of protein-DNA interactions.

Protein-DNA interactions provide fundamental control mechanisms over biologically essential processes such as DNA replication, transcription, and repair. However, many details of these mechanisms still remain unclear. Atomic force microscopy (AFM) analyses provide unique and important structural and functional information on such protein-DNA interactions at the level of the individual molecules.