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Nucleic Acids Res. 2015 Mar 11;43(5):2716-29. doi: 10.1093/nar/gkv139. Epub 2015 Feb 24.

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

Author information

1
Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.
2
Department of Biochemistry and Molecular Biology and Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
3
Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany ingrid.tessmer@virchow.uni-wuerzburg.de.

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.

PMID:
25712093
PMCID:
PMC4357730
DOI:
10.1093/nar/gkv139
[Indexed for MEDLINE]
Free PMC Article

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