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Tautomerization-Dependent Recognition and Excision of Oxidation Damage in Base-Excision DNA Repair

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Tautomerization-Dependent Recognition and Excision of Oxidation Damage in Base-Excision DNA Repair Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair Chenxu Zhua,1, Lining Lua,1, Jun Zhangb,c,1, Zongwei Yuea, Jinghui Songa, Shuai Zonga, Menghao Liua,d, Olivia Stoviceke, Yi Qin Gaob,c,2, and Chengqi Yia,d,f,2 aState Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China; bInstitute of Theoretical and Computational Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China; cBiodynamic Optical Imaging Center, Peking University, Beijing 100871, China; dPeking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China; eUniversity of Chicago, Chicago, IL 60637; and fSynthetic and Functional Biomolecules Center, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Edited by Suse Broyde, New York University, New York, NY, and accepted by Editorial Board Member Dinshaw J. Patel June 1, 2016 (received for review March 29, 2016) NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mamma- (FapyA and FapyG), spiroiminodihydantoin (Sp), and guanidino- lian genome against oxidized DNA bases. As the first enzymes in hydantoin (Gh) (23–27). Among these lesions, Tg is the most the base-excision repair pathway, glycosylases must recognize common pyrimidine base modification produced under oxidative the cognate substrates and catalyze their excision. Here we stress and ionizing radiation (28)—arising from oxidation of thy- present crystal structures of human NEIL1 bound to a range of mine and 5-methylcytosine—and is also a preferred substrate of duplex DNA. Together with computational and biochemical NEIL1 (3). When left unrepaired, Tg severely blocks replicative analyses, our results suggest that NEIL1 promotes tautomerization polymerases in continued DNA synthesis and elicits cytotoxic lethal of thymine glycol (Tg)—a preferred substrate—for optimal binding in its active site. Moreover, this tautomerization event also facili- consequences (29). Interestingly, Tg can be differentially processed tates NEIL1-catalyzed Tg excision. To our knowledge, the present by two naturally existing NEIL1 forms: one with a Lys at position example represents the first documented case of enzyme- 242, which is encoded in the genome, and the other with an Arg242 promoted tautomerization for efficient substrate recognition and residue, due to RNA editing of NEIL1 pre-mRNA; the former BIOCHEMISTRY catalysis in an enzyme-catalyzed reaction. removes Tg from duplex DNA ∼30 times faster than the latter (30). Subsequent binding analysis using synthetic Tg analogs shows base-excision repair | substrate recognition | enzyme catalysis | that binding affinities are similar for the two NEIL1 forms, hinting glycosylase | QM/MM that the editing effect is beyond substrate affinity (31). Although the crystal structure of NEIL1 protein is determined (32), a com- NA oxidation damage can be induced by both endogenous plex structure of NEIL1 bound to duplex DNA has not been Dand environmental reactive oxygen species. Such oxidized reported. Recent studies have determined several complex struc- DNA bases are primarily recognized and removed by the base- tures of MvNei1 (mimivirus Nei1), a viral ortholog of human excision repair (BER) pathway, which is initiated by a lesion- NEIL1 (33, 34). However, MvNei1 and NEIL1 have apparent specific DNA glycosylase (1–4). Based on sequence homology differences in their 3D structures and enzymatic properties (5). and structural motifs, glycosylases that cleave oxidation damage Hence, structures of human NEIL1 with bound substrates are are grouped into two families: the helix–hairpin–helix (HhH) family and the Fpg/Nei family (5, 6); the latter is named after the prototypical bacterial members formamidopyrimidine DNA Significance glycosylase (Fpg) and endonuclease eight (Nei). NEIL1 (Nei-like 1) is one such Fpg/Nei family glycosylase that Oxidative DNA damage can be cytotoxic or mutagenic to cells. guards the mammalian genome against oxidation damage (7–10). NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the NEIL1 is bifunctional in that it catalyzes both the hydrolysis of the mammalian genome against various oxidized DNA bases; yet N-glycosylic bond linking a base to a deoxyribose (glycosylase how NEIL1 recognizes and catalyzes the removal of its sub- activity) and the subsequent cleavage of the DNA 3′ to the newly strates remains poorly understood. Here we integrate crystal created apurinic/apyrimidinic site (lyase activity) (7–10). The structures of a NEIL1/double-stranded DNA complex, compu- N terminus contains the glycosylase domain of NEIL1, and the tational simulations, and biochemical analyses and show that C terminus is intrinsically disordered (8, 11). Whereas the C ter- NEIL1 promotes tautomerization of thymine glycol—a cognate minus is dispensable for both glycosylase and lyase activities substrate—for efficient substrate recognition and excision. in vitro, it interacts with many proteins in vivo and is required for Such tautomerism-dependent substrate recognition and catal- efficient DNA repair activity inside the cells (12, 13). NEIL1 ysis is, to the best of our knowledge, reported for the first time is also unique among the three human NEIL proteins in that in an enzyme-catalyzed reaction. it is increased in an S-phase–specific manner and carries out prereplicative repair of oxidized bases in the human genome (8, Author contributions: C.Z., L.L., J.Z., J.S., Y.Q.G., and C.Y. designed research; C.Z., L.L., J.Z., Z.Y., J.S., S.Z., and M.L. performed research; C.Z., L.L., J.Z., Z.Y., J.S., S.Z., Y.Q.G., and C.Y. 12). Moreover, increasing literature has further emphasized the analyzed data; and C.Z., L.L., J.Z., J.S., O.S., Y.Q.G., and C.Y. wrote the paper. ’ importance of NEIL1 s cellular repair activity, as NEIL1 defi- The authors declare no conflict of interest. ciency has led to multiple abnormalities and is associated with – This article is a PNAS Direct Submission. S.B. is a guest editor invited by the Editorial severe human diseases, including cancer (14 19). Additionally, Board. emerging evidence has also implicated a role of NEIL1 in active Data deposition: The atomic coordinates and structure factors have been deposited in the DNA demethylation (20–22). Protein Data Bank, www.pdb.org (PDB ID codes 5ITQ, 5ITT, 5ITR, 5ITU, 5ITY, and 5ITX). NEIL1 is capable of removing a wide array of oxidized py- 1C.Z., L.L., and J.Z. contributed equally to this work. rimidines and purines; representative substrates of extensive 2To whom correspondence may be addressed. Email: [email protected] or chengqi.yi@ investigations include thymine glycol (Tg), 5-hydroxyuracil (5- pku.edu.cn. OHU), 5-hydroxycytosine (5-OHC), dihydrothymine (DHT), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and dihydrouracil (DHU), as well as the formamidopyridines 1073/pnas.1604591113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1604591113 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 desired to understand the mechanism of substrate recognition and excision by NEIL1. Here we report the structures of human NEIL1 bound to dsDNA containing either a tetrahydrofuran (THF), a stable abasic site analog, or a Tg. Our structures show a flexible loop that adopts distinct conformations in the free protein, THF-, and Tg-bound structures. Combining crystallographic observations with quantum mechanics/molecular mechanics (QM/MM) sim- ulations and biochemical investigations, we reveal that NEIL1 promotes tautomeric shifts of Tg in its active site, so as to allow optimal substrate binding. Moreover, the enzyme-promoted tautomerization also facilitates the NEIL1-mediated Tg excision reaction. Such tautomerism-dependent substrate recognition and catalysis is reported, to the best of our knowledge, for the first time in base-excision DNA repair. Results Crystal Structures of NEIL1/dsDNA Complexes. We first identified a truncated NEIL1 lacking 95 amino acids at the C-terminal do- main (CTD) for use in complex structure determination. This NEIL1CΔ95 truncation retained similar in vitro repair activities compared with the WT protein; its free protein structure (solved to 1.48 Å) further supported that additional truncations at the CTD have minimal impact on the structure of NEIL1’s glycosylase domain (SI Appendix, Fig. S1) [with rmsd ∼0.23 Å to the previously determined NEIL1CΔ56 structure; Protein Data Bank (PDB) 1TDH]. Hence, NEIL1CΔ95 was used for struc- tural characterizations of NEIL1/dsDNA complexes. Fig. 1. Crystal structures of NEIL1 bound to dsDNA containing THF and Tg, We solved the structures of NEIL1 bound to dsDNA containing respectively. (A) Overall view of NEIL1 bound to a THF-containing duplex, with the flipped THF shown in purple. (B) Accommodation of THF in the either a THF (a stable abasic site analog) or a Tg (a preferred active site of NEIL1. Density map (2Fobs − Fcal) of THF is shown. Hydrogen substrate of NEIL1) (Fig. 1). For the THF-bound complexes, we bonds are shown in green dashed lines. The red dashed line shows the dis- used both the WT NEIL1 and a catalytically deficient NEIL1 tance between the α-amino group of Pro2 and C1 atom of THF. (C) Overall variant (P2G); an overlay of the two structures showed that this view of NEIL1 bound to a Tg-containing duplex, with the flipped Tg shown in mutation introduced minimal perturbation to the recognition of purple. (D) Overlay of the apo and THF-bound NEIL1 structures, highlighting THF (SI Appendix,Fig.S2A and B). Thus, this NEIL1 P2G the conformational change of Arg242. In the Tg-bound structure, Arg242 in mutation was further used for the Tg-bound structures. the flexible lesion recognition loop flips over, and the polar side chain of Arg242 points to the Tg base. Tyr244 resides approximately in the same NEIL1 Uses a Flexible Lesion Recognition Loop to Allow Substrate location in both apo (gray) and Tg-bound (green) structures.
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