Abstract
Base excision repair is the dominant DNA repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic potential. Detection and excision of these base lesions is achieved by DNA glycosylases. To investigate the remarkably high efficiency in target site search and recognition by these enzymes, we applied single molecule atomic force microscopy (AFM) imaging to a range of glycosylases with structurally different target lesions. Using a novel, automated, unbiased, high-throughput analysis approach, we were able to resolve subtly different conformational states of these glycosylases during DNA lesion search. Our results lend support to a model of enhanced lesion search efficiency through initial lesion detection based on altered mechanical properties at lesions. Furthermore, its enhanced sensitivity and easy applicability also to other systems recommend our novel analysis tool for investigations of diverse, fundamental biological interactions.
Highlights
Base excision repair (BER) is the dominant repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic p otential[1,2,3]
Using atomic force microscopy (AFM) imaging, we have previously shown a dynamic equilibrium between an interrogation complex (IC) conformation and a mildly bent species representing the search complex (SC) conformation for two glycosylases, human thymine DNA glycosylase and human oxo-guanine glycosylase bound to undamaged DNA
We suggested an initial lesion detection strategy for human thymine DNA glycosylase (hTDG) and hOGG1, in which structural and/or mechanical properties of their target lesions serve as a pre-selection criterion for lesion probing[6]
Summary
Base excision repair (BER) is the dominant repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic p otential[1,2,3]. Using atomic force microscopy (AFM) imaging, we have previously shown a dynamic equilibrium between an interrogation complex (IC) conformation (in which the glycosylase attempts to flip the target base into its catalytic pocket by strongly bending the DNA) and a mildly bent species representing the search complex (SC) conformation for two glycosylases, human thymine DNA glycosylase (hTDG) and human oxo-guanine glycosylase (hOGG1) bound to undamaged DNA. A high throughput analysis method that semiautomatically determines DNA lengths and protein-DNA complex volumes has previously been made available to AFM users[19] This approach allows for the semi-automated measurement of DNA bending at manually selected sites in the DNA. The software is available at Open Science Framework at https://osf.io/yhwuc/
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