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Exonuclease Activity and Removal of Bulky Insight into mechanisms of 3′-5′ exonuclease activity PNAS PLUS and removal of bulky 8,5′-cyclopurine adducts by apurinic/apyrimidinic endonucleases Abdelghani Mazouzia, Armelle Vigourouxb, Bulat Aikesheva,c, Philip J. Brooksd,e, Murat K. Saparbaeva, Solange Morerab,1, and Alexander A. Ishchenkoa,1 aUniversité Paris-Sud, Laboratoire “Stabilité Génétique et Oncogenèse”, Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche 8200, Institut de Cancérologie Gustave-Roussy, F-94805 Villejuif Cedex, France; bLaboratoire d’Enzymologie et Biochimie Structurales, CNRS, F-91198 Gif-sur-Yvette Cedex, France; cL.N. Gumilev Eurasian National University, Astana, Republic of Kazakhstan, 010008; and dLaboratory of Neurogenetics and Division of Metabolism and Health Effects, National Institute on Alcohol Abuse and Alcoholism, eOffice of Rare Diseases Research, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892 Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved June 28, 2013 (received for review March 19, 2013) 8,5′-cyclo-2’-deoxyadenosine (cdA) and 8,5′-cyclo-2’-deoxyguano- compared with regular dA, implying that this base lesion would sine generated in DNA by both endogenous oxidative stress and be resistant to DNA glycosylase action (3). ionizing radiation are helix-distorting lesions and strong blocks for The cdA adducts in DNA are a strong block to various DNA DNA replication and transcription. In duplex DNA, these lesions are polymerases, such as T7, δ, and η (4). Interestingly, translesion repaired in the nucleotide excision repair (NER) pathway. How- DNA polymerase η can perform lesion bypass synthesis on the ever, lesions at DNA strand breaks are most likely poor substrates R-cdA but not on S-cdA (5). Both diastereomers of cdA also for NER. Here we report that the apurinic/apyrimidinic (AP) endo- inhibit DNA transcription by blocking primer extension by T7 nucleases—Escherichia coli Xth and human APE1—can remove 5′S DNA polymerase, and S-cdA inhibits binding of the TATA box cdA (S-cdA) at 3′ termini of duplex DNA. In contrast, E. coli Nfo and protein in vitro and strongly reduces gene expression in vivo (6). yeast Apn1 are unable to carry out this reaction. None of these In addition, in vivo human RNA polymerase II generates mutated enzymes can remove S-cdA adduct located at 1 or more nt away RNA transcripts when using DNA template containing S-cdA (7). from the 3′ end. To understand the structural basis of 3′ repair Given the strong genotoxic effect of cdA adducts on DNA me- activity, we determined a high-resolution crystal structure of E. coli tabolism, cells should have a repair mechanism to remove these Nfo-H69A mutant bound to a duplex DNA containing an α-anomeric helix-distorting DNA adducts. Indeed, it was shown that the nu- 2′-deoxyadenosine:T base pair. Surprisingly, the structure reveals a cleotide excision repair (NER) pathway can remove cdA adducts bound nucleotide incision repair (NIR) product with an abortive 3′- with efficiency comparable to that of T = T cyclobutane dimers terminal dC close to the scissile position in the enzyme active site, and exhibits higher activity in excising the R-isomer (4, 8). In providing insight into the mechanism for Nfo-catalyzed 3′→5′ exo- agreement with the biochemical data, it was shown that cdPu ′ S BIOCHEMISTRY nuclease function and its inhibition by 3 -terminal -cdA residue. adducts accumulate in keratinocytes from xeroderma pigmento- ′ This structure was used as a template to model 3 -terminal residues sum group C and Cockayne syndrome (CS) group A patients ex- in the APE1 active site and to explain biochemical data on APE1- posed to X-rays and potassium bromate (KBrO ) (9, 10) and also ′ 3 catalyzed 3 repair activities. We propose that Xth and APE1 may in organs of CS group B knockout mice (11). Importantly, cdA act as a complementary repair pathway to NER to remove S-cdA adducts from 3′ DNA termini in E. coli and human cells, respectively. Significance oxidative DNA damage | endonuclease IV | DNA glycosylase | base excision repair | damage specific endonuclease Oxidative DNA damage has been postulated to play an im- portant role in human neurodegenerative disorders and cancer. 8,5′-cyclo-2′-deoxyadenosine (cdA) is generated in DNA by xidative damage to DNA caused by reactive oxygen species hydroxyl radical attack and strongly blocks DNA replication Ois believed to be a major type of endogenous cellular damage. and transcription. Here we demonstrate that cdA adducts at If unrepaired, the damage will tend to accumulate and lead to 3′ termini of DNA can be removed by 3′-5′ exonuclease activity premature aging, neurodegenerative disorders, and cancer (1). of the apurinic/apyrimidinic (AP) endonucleases: Escherichia More than 80 different oxidative modifications of DNA bases coli Xth and human APE1. The crystal structure of bacterial AP and sugar backbone have been identified to date (2). Diaste- endonuclease in complex with DNA duplex provides insight reoisomeric (5′S)- and (5′R)-8,5′-cyclo-2′-deoxyadenosine (cdA) into the mechanism of this activity. This new repair function and 8,5′-cyclo-2′-deoxyguanosine (cdG) are generated by endog- provides an alternative pathway to counteract genotoxic effect enous oxidative stress and ionizing radiation among other oxidized of helix-distorting DNA lesions. bases (Fig. 1A). 8,5′-cyclo-2′-deoxypurines (cdPu) are generated by ′ Author contributions: A.M., A.V., M.K.S., S.M., and A.A.I. designed research; A.M., A.V., hydroxyl radical attack at C5 sugar by H-abstraction resulting in B.A., S.M., and A.A.I. performed research; P.J.B. contributed new reagents/analytic tools; formation of C5′-centered sugar radical, which then reacts in the A.M., B.A., P.J.B., M.K.S., S.M., and A.A.I. analyzed data; and P.J.B., M.K.S., S.M., and A.A.I. absence of oxygen with the C8 of the purine. Subsequent oxidation wrote the paper. of the resulting N7-centered radical leads to intramolecular cy- The authors declare no conflict of interest. clization with the formation of a covalent bond between the C5′- This article is a PNAS Direct Submission. and C8-positions of the purine nucleoside. When present in DNA Freely available online through the PNAS open access option. duplex cdA causes large changes in backbone torsion angles, Data deposition: The atomic coordinates and structure factors of H69A Endo IV:DNA have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 4K1G). which leads to weakening of base pair hydrogen bonds and 1To whom correspondence may be addressed. E-mail: [email protected] or strong perturbations of the helix conformation near the lesion [email protected]. for both diastereoisomers. Interestingly, the glycosidic bond in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. S-cdA is approximately 40-fold more resistant to acid hydrolysis 1073/pnas.1305281110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1305281110 PNAS | Published online July 29, 2013 | E3071–E3080 Downloaded by guest on September 29, 2021 coupled NER may participate in cleansing single-strand breaks from this 3′ adduct (13). Therefore, it is unlikely that NER could be able to efficiently remove cdPu located at 3′ termini of a sin- gle-strand break. Recently it has been demonstrated that 5′R and 5′S isomers of cdATP could be incorporated with low efficiency by replicative DNA polymerases and then inhibit further DNA synthesis, thus potentially generating gapped DNA with a cdA adduct at the 3′ end (14). Furthermore, in the absence of ion- izing radiation and/or drugs, cdPu could arise at 3′ termini as a result of 3′→5′ exonuclease degradation of DNA, for example by TREX1 (5). The majority of oxidatively damaged DNA bases are sub- strates for two overlapping pathways: DNA glycosylase-initiated base excision repair (BER) and apurinic/apyrimidinic (AP) en- donuclease-mediated nucleotide incision repair (NIR) (15). In the NIR pathway, an AP endonuclease makes an incision 5′ to a damaged nucleotide and then extends the resulting single-strand break to a gap by a nonspecific3′→5′ exonuclease activity (16, 17). AP endonucleases are multifunctional DNA repair enzymes that possess AP site nicking, 3′ repair diesterase, NIR, and 3′→5′ exonuclease activities and are divided into two distinct families based on amino acid sequence identity to either Escherichia coli exonuclease III (Xth) or endonuclease IV (Nfo) (18). Human APE1 is homologous to Xth, whereas Saccharomyces cerevisiae Apn1 is homologous to Nfo. Previously it was shown that AP endonuclease-catalyzed 3′→5′ exonuclease activity could serve as a3′ editing function for removing mismatched and oxidized bases at 3′ termini of DNA duplex (19–21). However, the de- tailed mechanisms for those 3′ editing repair activities are not yet clearly understood. Although Xth and Nfo AP endonuclease families share common DNA substrate specificities, they are distinguished by their modes of DNA damage recognition. In- deed, cocrystal structures of Nfo bound to an AP site analog, tetrahydrofuran (THF), showed that the enzyme drastically dis- torts the DNA helix by ∼90° bending and flips out not only the target AP site but also its opposing nucleotide out of the DNA base stack (22, 23). Interestingly, the Nfo active site pocket sterically excludes binding of normal β-configuration nucleotides, but it can fit α-anomeric nucleotides. In contrast, cocrystal struc- tures of APE1 bound to abasic site-containing DNA show that the enzyme kinks the DNA helix by only 35° and binds a flipped-out Fig. 1. Repair of 3′-blocking bulky adducts by AP endonucleases-catalyzed AP site in a pocket that excludes DNA bases, whereas the op- ′→ ′ ′ ′ posite base remains stacked in the duplex (24).
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