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Uracil in Duplex DNA Is a Substrate for the Nucleotide Incision Repair

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Uracil in Duplex DNA Is a Substrate for the Nucleotide Incision Repair Uracil in duplex DNA is a substrate for the nucleotide PNAS PLUS incision repair pathway in human cells Paulina Proroka,b, Doria Alilib, Christine Saint-Pierrec, Didier Gasparuttoc, Dmitry O. Zharkovd,e, Alexander A. Ishchenkob, Barbara Tudeka,f,1, and Murat K. Saparbaevb,1 aInstitute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland; bGroupe Réparation de l’ADN, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8200, Université Paris-Sud, Institut de Cancérologie Gustave Roussy, F-94805 Villejuif Cedex, France; cLaboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique/Unité Mixte de Recherche E3 Commissariat à l’Energie Atomique–Université Joseph Fourier, Institut Nanosciences et Cryogénie, F-38054 Grenoble, France; dSiberian Branch of the Russian Academy of Sciences, Institute of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia; eDepartment of Natural Sciences, Novosibirsk State University, Novosibirsk 630090, Russia; and fInstitute of Genetics and Biotechnology, University of Warsaw, 00-927, Warsaw, Poland Edited by James E. Cleaver, University of California, San Francisco, CA, and approved August 13, 2013 (received for review March 25, 2013) Spontaneous hydrolytic deamination of cytosine to uracil (U) in and leave an apurinic/apyrimidinic (AP) site, which in turn is DNA is a constant source of genome instability in cells. This cleaved by an AP endonuclease to generate a single-stranded mutagenic process is greatly enhanced at high temperatures and break flankedwith3′-hydroxyl (3′-OH) and 5′-deoxyribose in single-stranded DNA. If not repaired, these uracil residues give phosphate (5′-dRp) groups. Next, using the 3′-OH terminus, a rise to C→T transitions, which are the most common spontaneous DNA polymerase initiates repair synthesis to incorporate reg- mutations occurring in living organisms and are frequently found ular nucleotides and to remove the 5′-dRp group. Finally, a DNA in human tumors. In the majority of species, uracil residues are ligase seals the nick to restore the DNA duplex integrity (8, 9). removed from DNA by specific uracil-DNA glycosylases in the base DNA glycosylases acting on genomic uracil are found in all living excision repair pathway. Alternatively, in certain archaeal organ- organisms and belong to the uracil-DNA glycosylase (UDG) isms, uracil residues are eliminated by apurinic/apyrimidinic (AP) structural superfamily, which consists of five families (10, 11). endonucleases in the nucleotide incision repair pathway. Here, we Family 1 UNGs, highly conserved in prokaryotes and eukaryotes, characterized the substrate specificity of the major human AP en- fi are the most ef cient enzymes for the removal of uracil BIOCHEMISTRY donuclease 1, APE1, toward U in duplex DNA. APE1 cleaves oligo- from DNA. · nucleotide duplexes containing a single U G base pair; this activity Intriguingly, certain Archaea and pupating insects contain no depends strongly on the sequence context and the base opposite uracil-DNA glycosylases (12, 13). Complete genome sequences to U. The apparent kinetic parameters of the reactions show that of several termophilic and mesophilic archaean species did not fi APE1 has high af nity for DNA containing U but cleaves the DNA reveal homology to any of the five UDG families, raising duplex at an extremely low rate. MALDI-TOF MS analysis of the a question of how these organisms deal with the uracil threat to reaction products demonstrated that APE1-catalyzed cleavage of genome stability (14, 15). Using a biochemical approach, Fritz aU·G duplex generates the expected DNA fragments containing and coworkers have identified the Mth212 protein, a homolog of a5′-terminal deoxyuridine monophosphate. The fact that U in the Escherichia coli AP endonuclease Xth, as a DNA uridine duplex DNA is recognized and cleaved by APE1 in vitro suggests endonuclease in Methanothermobacter thermautotrophicus ΔH that this property of the exonuclease III family of AP endonu- (12). It has been shown that Mth212 can cleave 5′ to AP sites and cleases is remarkably conserved from Archaea to humans. We pro- deoxyuridines (dU) in duplex DNA and also possesses an effi- pose that nucleotide incision repair may act as a backup pathway ′→ ′ to base excision repair to remove uracils arising from cytosine cient 3 5 exonuclease activity, indicating that this archaeal deamination. protein is a true AP endonuclease. Furthermore, DNA glyco- sylase-independent repair of uracil residues was reconstituted spontaneous DNA base deamination | alternative excision repair | evolution Significance pontaneous hydrolytic deamination of cytosine to uracil Sgenerates a highly mutagenic DNA base lesion. About 70– Hydrolytic deamination of cytosine to uracil generates a highly 200 genomic cytosine bases per day are converted to uracils in mutagenic DNA base lesion and is considered one of the major every human cell (1, 2). Importantly, the rate of cytosine de- sources of spontaneous mutation in living organisms. We re- amination is greatly increased at elevated temperatures and in port that the major human apurinic/apyrimidinic (AP) endo- single-stranded DNA. If not repaired, the uracil will pair with nuclease, APE1, is a deoxyuridine endonuclease and can remove adenine during DNA replication and will inevitably produce uracil residues in the DNA glycosylase-independent nucleotide aC·G→T·A transition mutation. Indeed, C→T transitions, likely incision repair pathway. This new repair function of AP endo- arising through cytosine damage, are the most frequently oc- nucleases is evolutionarily conserved in Archaea and humans, curring base substitutions observed in living organisms (3–5). In pointing to a possible evolutionary origin of the DNA repair addition, uracil in DNA occurs from incorporation of dUMP mechanisms for spontaneous damage to DNA in a common instead of TMP from the nucleotide pool; this process results in ancestor to all living forms. U·A pairs that are not mutagenic but can be lethal for cells when Author contributions: P.P., D.G., A.A.I., B.T., and M.K.S. designed research; P.P., D.A., C.S.-P., accumulated at a high level in genomic DNA (6, 7). To coun- and A.A.I. performed research; C.S.-P. and D.G. contributed new reagents/analytic tools; teract genotoxic effects of uracil in DNA, organisms from all P.P., D.A., C.S.-P., D.G., D.O.Z., A.A.I., B.T., and M.K.S. analyzed data; and D.O.Z., B.T., and major domains of life (Archaea, Bacteria, and Eukarya, as well M.K.S. wrote the paper. as some eukaryotic viruses) use the base excision repair (BER) The authors declare no conflict of interest. pathway, which is mediated by DNA glycosylases. Uracil removal This article is a PNAS Direct Submission. in BER is initiated by highly specific and efficient uracil-DNA N- Freely available online through the PNAS open access option. glycosylases (UNGs) that excise uracil from DNA by hydrolyzing 1To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. the glycosidic bond between the abnormal base and the sugar. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. UNGs are monofunctional DNA glycosylases that excise uracil 1073/pnas.1305624110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1305624110 PNAS Early Edition | 1of9 Downloaded by guest on October 1, 2021 using four purified proteins of M. thermautotrophicus: Mth212, DNA glycosylase-catalyzed excision of U in the 3′-labeled U·G DNA polymerase B, 5′-flap endonuclease, and DNA ligase (16). duplex should generate a 20-mer cleavage fragment that is shorter Interestingly, Xth family AP endonucleases other than Mth212, than the NIR product by one nucleotide, the dUMP (17). To including E. coli Xth, Methanosarcina mazei Mm3148, and hu- discriminate between BER and NIR activities, we generated man AP endonuclease 1 (APE1), do not exhibit DNA uridine the 20-mer BER product cleaving the U·G duplex with the endonuclease activity under the experimental conditions used to mismatch-specific uracil/thymine DNA glycosylase (TDG) and measure it (12). APE1 under the BER+Mg2+ reaction conditions. TDG is known Previously, we have proposed that the classic DNA glyco- toexciseUwithhighefficiency from mismatched U·G but not sylase-initiated BER pathway raises theoretical problems for the from U·A or single-stranded DNA (33, 34). As shown in Fig. 1A, efficient repair of DNA damage because it generates highly a prolonged incubation (up to 180 min at 37 °C) of U·G with ′ genotoxic intermediates such as AP sites and/or blocked 3 -termini APE1 under NIR conditions led to formation of a specificcleav- that must be eliminated by additional steps before initiating age product that migrated slower than the 20-mer fragment gen- the repair synthesis. Work in our laboratory, together with other erated by TDG (lanes 4, 6, 8, 10, 12, and 14 versus lane 2). This fi observations, had identi ed an alternative to the classic BER, cleavage pattern indicates that APE1 incises 5′ next to dU and the nucleotide incision repair (NIR) pathway, in which an AP generates a 21-mer cleavage fragment containing the 5′-dangling endonuclease makes an incision 5′ to a damaged base in a DNA ′ dUMP residue. As expected, no detectable activity of APE1 on the glycosylase-independent manner, resulting in a free 3 -OH group U·G duplex was observed in the BER+Mg2+ reaction buffer suitable for DNA polymerases and a 5′-dangling damaged nu- — E. coli (lanes 3, 5, 7, 9, 11, and 13). Importantly, despite APE1 sharing cleotide (17). The NIR endonucleases including Nfo, common substrate specificity with endonuclease IV family NIR Saccharomyces cerevisae Apn1, and human APE1—can directly endonucleases, we observed no specific cleavage when the 3′- cleave DNA duplexes within a strand containing α-anomeric labeled U·G duplex was incubated with Nfo or Apn1 (Fig.
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