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. 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.

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. Notably, for both hTDG and a second glycosylase, hOGG1, which recognizes structurally different 8-oxoguanine lesions, the conformation of the DNA in the SC mirrors innate structural properties of their respective target sites. In the IC, the DNA is sharply bent, as seen in crystal structures of hTDG lesion recognition complexes, which likely supports the base flipping required for lesion identification. Our results support a potentially general concept of sculpting of glycosylases to their targets, allowing them to exploit the energetic cost of DNA bending for initial lesion sensing, coupled with continuous (extrahelical) base interrogation during lesion search by DNA glycosylases.

A conformational switch in collybistin determines the differentiation of inhibitory postsynapses.

The formation of neuronal synapses and the dynamic regulation of their efficacy depend on the assembly of the postsynaptic neurotransmitter receptor apparatus. Receptor recruitment to inhibitory GABAergic and glycinergic synapses is controlled by the scaffold protein gephyrin and the adaptor protein collybistin. We derived new insights into the structure of collybistin and used these to design biochemical, cell biological, and genetic analyses of collybistin function. Our data define a collybistin-based protein interaction network that controls the gephyrin content of inhibitory postsynapses. Within this network, collybistin can adopt open/active and closed/inactive conformations to act as a switchable adaptor that links gephyrin to plasma membrane phosphoinositides. This function of collybistin is regulated by binding of the adhesion protein neuroligin-2, which stabilizes the open/active conformation of collybistin at the postsynaptic plasma membrane by competing with an intramolecular interaction in collybistin that favors the closed/inactive conformation. By linking trans-synaptic neuroligin-dependent adhesion and phosphoinositide signaling with gephyrin recruitment, the collybistin-based regulatory switch mechanism represents an integrating regulatory node in the formation and function of inhibitory postsynapses.

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. It has been proposed that the 5'-3' helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a "final" verification state, which may then trigger the recruitment of further NER proteins.

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. The high sensitivity of the method with topographical visualization of all sample components also demands for extremely clean and pure materials. Here, we provide an overview of molecular biology-based approaches to produce DNA substrates for AFM imaging as well as other types of experiments, such as optical or magnetic tweezers, that profit from controllable substrate properties in long DNA fragments. We present detailed strategies to produce different types of motifs in DNA that are frequently employed targets of protein interactions. Importantly, the presented preparation techniques imply exact knowledge of the location of the introduced specific target sites within the DNA fragments, allowing for a distinction between specific and non-specific protein-DNA interactions in the AFM images and for separate conformational analyses of the different types of protein-DNA complexes.