Abstract
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.
Highlights
Base excision repair (BER) is the first-line defense to protect the genome from detrimental effects of cytotoxic and mutagenic DNA base oxidation, deamination and alkylation [1]
Focusing on the specific example of the human thymine DNA glycosylase, we address the question of DNA lesion search strategies of DNA glycosylases by atomic force microscopy (AFM) imaging. hTDG belongs to the UDG protein superfamily [29] and is involved in BER as well as epigenetic gene regulation being responsible for active DNA demethylation [30,31,32]
We introduced a single target site for hTDG, either about finding their target sites (a G):T or a G:U base mismatch in a CpG context, into long DNA fragments (549 bp)
Summary
Base excision repair (BER) is the first-line defense to protect the genome from detrimental effects of cytotoxic and mutagenic DNA base oxidation, deamination and alkylation [1]. Initial detection and removal of these DNA lesions within the huge excess of normal bases is achieved by DNA glycosylases. The question of how BER enzymes detect their target sites within the huge excess of undamaged bases remains largely unresolved. The base-sugar (N-glycosidic) bond of the everted base is cleaved, creating an abasic site in the DNA. Because these sites are highly susceptible to ssDNA breaks [9,10], abasic sites are protected by the glycosylase after base excision until further processing either by an apyrimidinic/apurinic endonuclease (for monofunctional glycosylases) or via an additional intrinsic apyrimidinic/apurinic lyase activity of the glycosylase (for bifunctional glycosylases). Further downstream BER factors are recruited to complete the repair reaction and genomic integrity is regained [11]
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