2716–2729 Nucleic Acids Research, 2015, Vol. 43, No. 5 Published online 24 February 2015 doi: 10.1093/nar/gkv139 Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging Claudia N. Buechner1,AtanuMaiti2, Alexander C. Drohat2 and Ingrid Tessmer1,* 1Rudolf Virchow Center for Experimental Biomedicine, University of Wurzburg,¨ Wurzburg,¨ Germany and 2Department of Biochemistry and Molecular Biology and Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, USA Received December 03, 2014; Revised February 09, 2015; Accepted February 10, 2015 ABSTRACT compromise between the needs to minimize the time for lesion search and to maximize the selectivity for removal The ability of DNA glycosylases to rapidly and effi- of their target base (2). Like other DNA binding proteins ciently detect lesions among a vast excess of non- (3,4), under the in vivo conditions of high DNA concen- damaged DNA bases is vitally important in base exci- tration, DNA repair enzymes have developed the ability to sion repair (BER). Here, we use single molecule imag- bind nonspecific DNA with moderate affinity (5–7). One- ing by atomic force microscopy (AFM) supported by dimensional diffusion in contact with undamaged DNA a 2-aminopurine fluorescence base flipping assay to (sliding) and three-dimensional transfer among DNA seg- study damage search by human thymine DNA gly- ments (hopping) may then facilitate rapid lesion detection cosylase (hTDG), which initiates BER of mutagenic (4–6,8). Final target identification requires contacts with and cytotoxic G:T and G:U mispairs in DNA. Our data residues in the enzyme active site achieved by flipping of reveal an equilibrium between two conformational the damaged base into an extrahelical state. In the catalytic step, the base-sugar (N-glycosidic) bond of the everted base states of hTDG–DNA complexes, assigned as search is cleaved, creating an abasic site in the DNA. Because these complex (SC) and interrogation complex (IC), both sites are highly susceptible to ssDNA breaks (9,10), abasic at target lesions and undamaged DNA sites. Notably, sites are protected by the glycosylase after base excision un- for both hTDG and a second glycosylase, hOGG1, til further processing either by an apyrimidinic/apurinic en- which recognizes structurally different 8-oxoguanine donuclease (for monofunctional glycosylases) or via an ad- lesions, the conformation of the DNA in the SC mir- ditional intrinsic apyrimidinic/apurinic lyase activity of the rors innate structural properties of their respective glycosylase (for bifunctional glycosylases). Finally, further target sites. In the IC, the DNA is sharply bent, as downstream BER factors are recruited to complete the re- seen in crystal structures of hTDG lesion recognition pair reaction and genomic integrity is regained (11). complexes, which likely supports the base flipping In the lesion recognition complex, many DNA glyco- required for lesion identification. Our results support sylases [e.g. uracil DNA glycosylase (UDG), hOGG1 or AlkA] (11) have been shown to display a high density of a potentially general concept of sculpting of glycosy- polar phosphodiester interactions with the damaged strand lases to their targets, allowing them to exploit the en- and only a few contacts with the undamaged strand, lead- ergetic cost of DNA bending for initial lesion sensing, ing to DNA backbone distortion (bending). The resulting coupled with continuous (extrahelical) base interro- protein-induced alternative DNA backbone conformations gation during lesion search by DNA glycosylases. and induced torsional stress on the target nucleotide have been shown to lower the energetic barrier for base flip- ping (12–14). Hereby, the lower stability of DNA at dam- INTRODUCTION aged compared to homoduplex sites (15,16) facilitates DNA Base excision repair (BER) is the first-line defense to protect bending and base flipping at target sites17 ( ,18) and en- the genome from detrimental effects of cytotoxic and muta- hances the probability for spontaneous base pair opening genic DNA base oxidation, deamination and alkylation (1). (base pair breathing). Currently, these processes are less well Initial detection and removal of these DNA lesions within understood for nonspecific DNA and the question whether the huge excess of normal bases is achieved by DNA glyco- DNA glycosylases bend and flip undamaged DNA during sylases. The question of how BER enzymes detect their tar- lesion search is still a matter of considerable debate (19,20). get sites within the huge excess of undamaged bases remains The mechanisms underlying enzymatic base flipping have largely unresolved. In general, DNA glycosylases have to been studied for several glycosylases (e.g. UDG, T4-Pdg, *To whom correspondence should be addressed. Tel: +49 931 3180425; Fax: +49 931 3187320; Email: [email protected] C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2015, Vol. 43, No. 5 2717 hOGG1, AlkA) with different techniques (2,17,21–24). A nition state, we use the catalytically inactive hTDG vari- major question is whether the proteins fulfill an active (in- ant N140A, which exhibits similar DNA binding affinity ducing base flipping) or passive (stabilizing the flipped state) as the wild-type (wt) enzyme, but only very minor, resid- role in nucleotide flipping. For example, for UNG glycosy- ual base excision activity for G:U and no detectable activ- lase, capture of transiently emerging bases (passive flipping) ity for G:T mispairs (40,41). This enables us to study in- has been demonstrated at nontarget sites (24–27). On the dividual hTDG–DNA complexes during lesion search and other hand, the glycosylase hOGG1 and its bacterial ho- detection in the absence of base excision. Our AFM data molog MutM were reported to exploit an active intrahelical analysis of protein-induced DNA bend angles revealed that base interrogation and extrusion mechanism (13,28). The hTDG adopts two clearly distinguishable complex confor- particular (active or passive) mechanism of base eversion mations both at damaged and undamaged DNA sites. Us- may hence vary for different glycosylases. Indeed, it seems ing mutational studies and a fluorescence-based nucleotide likely that not only base extrusion but the entire process of flipping assay, we confirmed an important role of the strictly target site search is optimized for the individual energetic conserved residue Arg275 in base flipping by TDG. Impor- requirements of the particular target sites of a specific gly- tantly, our data strongly suggest a lesion search mechanism cosylase. of TDG that involves an initial damage-sensing step, which Focusing on the specific example of the human thymine exploits mechanical properties of its target sites in DNA, DNA glycosylase (hTDG), we address the question of DNA coupled with (extrahelical) base interrogation. We present lesion search strategies of DNA glycosylases by atomic a model that summarizes our findings in the context of gen- force microscopy (AFM) imaging. hTDG belongs to the eral implications for DNA lesion search by DNA glycosy- UDG protein superfamily (29) and is involved in BER as lases. well as epigenetic gene regulation being responsible for ac- tive DNA demethylation (30–32). It shows a strong repair activity for uracil from G:U mispairs as well as for oxi- MATERIALS AND METHODS dized forms of methylated cytosine created by Tet enzymes, Enzymes including 5-formylcytosine and 5-carboxylcytosine (5fC, 5caC) (33,34). In addition, hTDG maintains the integrity hTDG wt and variants, N140A and R275A (all full length), of CpG sites by selective removal of thymine from G:T were expressed and purified as described (40). All experi- mismatches caused by deamination of 5-methylcytosine ments with the native TDG targets G:T and G:U were car- (5mC). Notably, hTDG efficiently avoids futile repair of ried out with the strongly incision impaired TDG-N140A undamaged DNA; the enzyme excises thymine, a normal variant (40), while wt or R275A variants [with residual base base, with 18 000-fold greater activity from G:T mispairs excision activity (40)] were incubated with the G:U analog F than from A:T pairs (35). Recent crystal structures of the G:U that cannot be processed by hTDG. Recombinant human 8-oxoguanine glycosylase (hOGG1) was obtained hTDG catalytic core domain (hTDGcat) showed the en- zyme in the lesion recognition state with the noncleavable from MyBioSource as N-terminally his-tagged and purified substrate analog 2-deoxy-2-flouroarabinouridine (UF)or protein with a concentration of 0.5 mg/ml as determined by 5caC, and the product complex with the deamination prod- Bradford assay. uct 5-hydroxymethyl uracil (5hmU) or an abasic sugar flipped into its active site31 ( ,36–38). Interestingly, in three DNA substrate preparation of these structures (31,36,38), the enzyme crystallized in a 2:1 complex with DNA, with one subunit bound to the Linear specific DNA substrates for AFM (549 bp) were pro- target lesion (specific complex) and one subunit bound to duced similarly as recently described (42,43). Briefly, DNA undamaged DNA in close vicinity (nonspecific complex). substrate preparation is based on the plasmid pUC19N Biochemical assays and kinetic studies confirmed dimer- (2729 base pairs, a gift from Samuel Wilson’s laboratory, ization (of the catalytic domain as well as the full length NIEHS, USA), which contains two restriction sites of the protein) under conditions of high and saturating [hTDG], nickase Nt.BstNBI (New England Biolabs, NEB) in close but showed that a monomer of hTDG was fully capable of vicinity. After nicking with Nt.BstNBI, the short ssDNA DNA lesion recognition and base excision (38,39).