J. Radiat. Res., 50, 19–26 (2009) Review

Generation, Biological Consequences and Repair Mechanisms of Deamination in DNA#

Shin-Ichiro YONEKURA, Nobuya NAKAMURA, Shuji YONEI and Qiu-Mei ZHANG-AKIYAMA*

Deamination of Cytosine// //Uracil-DNA Glycosylase/ dUTPase. Base moieties in DNA are spontaneously threatened by naturally occurring chemical reactions such as deamination, hydrolysis and oxidation. These DNA modifications have been considered to be major causes of cell death, mutations and cancer induction in organisms. Organisms have developed the DNA base excision repair pathway as a defense mechanism to protect them from these threats. DNA glycosy- lases, the key in the base excision repair pathway, are highly conserved in evolution. Uracil constantly occurs in DNA. Uracil in DNA arises by spontaneous deamination of cytosine to generate pro- mutagenic U:G mispairs. Uracil in DNA is also produced by the incorporation of dUMP during DNA replication. Uracil-DNA glycosylase (UNG) acts as a major repair enzyme that protects DNA from the deleterious consequences of uracil. The first UNG activity was discovered in E. coli in 1974. This was also the first discovery of base excision repair. The sequence encoded by the ung gene demonstrates that the E. coli UNG is highly conserved in viruses, bacteria, archaea, yeast, mice and humans. In this review, we will focus on central and recent findings on the generation, biological consequences and repair mechanisms of uracil in DNA and on the biological significance of uracil-DNA glycosylase.

ination by the complementary strand.1,4) Pro-mutagenic G:U GENERATION, BIOLOGICAL CONSEQUENCES mispairs cause G:C to A:T transition mutations, if not repaired OF CYTOSINE DEAMINATION prior to the next round of DNA replication.1,3–5,7) Deamination of cytosine in cyclobutane dimers is markedly faster than that 1,4,6) DNA reacts continually with H2O (hydrolysis) and reactive of cytosine in other sequences, and has been implicated in oxygen species, resulting in multiple spontaneous DNA UV- in both bacteria and mammalian cells.1,8,9) modifications.1–6) Four bases normally present in DNA contain Uracil also occurs in DNA through incorporation of exocyclic amino groups. The loss of these amino groups dUMP instead of dTMP by DNA polymerases during repli- (deamination) in cytosine, , and 5- cation.1,3–5,10,11) Incorporated dUMP forms U:A base pairs that methylcytosine occurs spontaneously and results in the are not directly mutagenic, but may be cytotoxic.1,3–5,12) conversion to uracil, , and , Substitution of thymine with uracil is a major source of respectively.3,4,6) The deamination of cytosine (Fig. 1) is endogenous abasic (AP) sites,12) which may mediate the cyto- estimated to result in 100–500 uracil residues per mammalian toxicity of uracil in DNA. dUTP is a normally occurring inter- genome per day.1,4–6) Cytosine deamination in single-stranded DNA is reported to be 200–300 times faster than that in double- stranded DNA.1,3–6) Single-stranded regions in replication forks and transcription bubbles are not protected from deam-

*Corresponding author: Phone: +075-753-4097, Fax: +075-753-4087, E-mail: [email protected] Department of Biological Sciences, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan. #Foot-note: Translated and modified from Radiat. Biol. Res. Comm. Vol. 42; 132–146 (2007, in Japanese). doi:10.1269/jrr.08080 Fig. 1. Deamination of the 4 position of cytosine generates uracil.

J. Radiat. Res., Vol. 50, No. 1 (2009); http://jrr.jstage.jst.go.jp 20 S.-I. Yonekura et al. mediate in nucleotide metabolism, but its level is kept low by U:A mispairs in DNA.1,3–5,18) Far more uracil residues than an efficient dUTPase (DUT). The pyrophosphatase activity of normal are incorporated into DNA in the dut mutant cells, dUTPase hydrolyzes dUTP to dUMP and PPi, which prevents as described above. The increased levels of uracil in DNA the incorporation of dUMP into DNA.1,3–5) Uracil incorpora- are also reduced by UNG. UNG does not possess associated tion into DNA is presumed to occur on an average of one per AP lyase activity, and the resultant AP sites are processed by 2,000–3,000 nucleotides in the dut mutant deficient in AP endonucleases.1,3–5,22) E. coli cells deficient in dUTPase dUTPase. Tye et al. (1978) reported that E. coli mutants defec- (dut) and exonuclease III (the major AP endonuclease in E. tive in both the dut and ung genes accumulate more uracil in coli, xthA) are therefore not viable.15) their DNA.10) Lari et al. (2006) determined the steady-state In E. coli, there are some uracil-removing enzymatic levels of uracil residues in DNA using the Ung-ARP assay.13) activities other than UNG. For example, mismatch-specific The uracil levels are 3,000–8,000/106 nucleotides in the ung uracil-DNA glycosylase (MUG) also removes uracil from dut mutant of E. coli, in contract to about 1/106 nucleotides in U:G mismatches.1,3-5) MUG has been thought to be a backup the wild-type strain (ung+ dut+).12,13) enzyme of UNG.3–5,23) MUG can act on mutagenic alkylated The frequency of spontaneous G:C to A:T transitions bases and 3,N4-ethenocytosine.23) significantly increases in E. coli and S. cerevisiae ung Endonuclease V (Nfi) in E. coli selectively cleaves mutants deficient in UNG.1,3–5,14–16) Increased spontaneous untreated single-stranded DNA and duplex DNA containing mutations in the ung mutants were also observed using λ uracil, hypoxanthine and mismatches.24–26) When the nfi bacteriophage.16) When λ phage is treated with , an mutant is treated with nitrous acid, it exhibits high frequencies agent known to cause cytosine deamination in DNA, the of A:T to G:C and G:C to A:T transition mutations.11,24–26) frequency of clear-plaque mutations is increased an Nitrous acid causes deamination of adenine and converts additional 20-fold when the phage is grown on E. coli ung adenine to hypoxanthine.26) The resulting deoxyinosine mutant rather than on ung+ cells.16) On the other hand, the (hypoxanthine deoxyribonucleoside) induces A:T to G:C frequency of bisulfite-induced mutations in E. coli ung transitions through pairing with cytosine, resulting in possible mutant is almost equal to that in the wild-type strain. The targets for Nfi.27) The Nfi enzyme was originally identified authors suggested that cellular uracil DNA glycosylase as a deoxyinosine 3’-endonuclease that cleaves DNA near might be inactivated by the bisulfite treatment.16) dIMP residues, mismatched bases, residues and AP sites.24–26) While nitrous acid causes deamination of cytosine URACIL-DNA GLYCOSYLASES IN BACTERIA, to generate uracil, it does not play the causative role in YEAST AND NEMATODE increased G:C to T:A transitions in the nfi mutant cells, because the ung mutant does not affect the transition fre- Endogenous DNA damage is mainly repaired by the base quency induced by nitrous acid. Schounten and Weiss excision repair pathway. The repair pathway is initiated by (1999) suggested a role for endonuclease V in the removal monofunctional DNA glycosylases that only remove the of deaminated guanine (i.e., xanthine) from DNA.26) altered base (e.g., uracil-DNA glycosylase) or bifunctional Bacteriophage PBS2 normally contains uracil instead of glycosylases that in addition to base removal incise the thymine in its DNA. Bacillus subtilis is the natural host for resulting apurinic/apyrimidinic (AP) site by an associated this bacteriophage. As indicated by the high level of UNG AP lyase activity (e.g., endonuclease III).1,3,4,17–19) The uracil activity present in extracts from B. subtilis, PBS2 would be N-glycosylase activity (UNG) of E. coli was the first DNA easily inactivated in B. subtilis. Following infection, the glycosylase to be discovered in a search for activities that phage induces the expression of PBS2 DNA encoded uracil- would repair uracil in DNA.20,21) UNG recognizes uracil in DNA glycosylase inhibitor (Ugi).28,29) Ugi forms a stable 1:1 DNA and removes it from the DNA backbone as the first complex with Ung and inactivates it. Ugi specifically inhib- step of the base excision repair pathway for uracil.20) The its the uracil-DNA glycosylase activity of UNG family-1 open reading frame of the ung gene in E. coli encodes a pro- .3–5,28,29) Therefore, Ugi is often used to examine tein of 229 amino acids. The purified enzyme from E. coil whether or not the biological effect of uracil-removing activ- hydrolyzes the N-glycosylic bond between uracil and deox- ities is due to the family 1 uracil-DNA glycosylase.3,30) yribose, thus releasing a free uracil and leaving an AP site In yeast, UNG homologs function in both the nucleus and in DNA.3–5) AP endonucleases recognize the resultant AP mitochondria. The ung-1 mutant of S. cerevisiae shows a site and cleave the phosphodiester backbone 5’ to the AP high frequency of spontaneous mutations in the nuclear site. This step is followed by DNA repair synthesis by DNA SUP4-o gene31) and mitochondrial petite loci, compared polymerases, and the DNA is finally rejoined by the with the isogenic wild-type cells.32) Deletion of the dut-1 action of DNA ligase.2–5) UNG of E. coli can remove roughly gene that encodes dUTPase is lethal in S. cerevisiae owing 800 uracil residues from DNA per minute and is present at to cell cycle arrest.4,33,34) However, the viability of the dut-1 about 300 per cell.4) strain is restored when the ung gene is disrupted simulta- UNG of E. coli removes uracil from both the U:G and neously or exogenous dTMP is supplied.4,33,34) Guillet et al.

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(2006) identified a viable allele (dut1-1) of the DUT gene in the family-1 UNG group. S. cerevisiae.34) The mutant shows growth delay and cell The ung-1 mutant of C. elegans36) is viable and fertile, and cycle abnormalities and exhibits a potent spontaneous muta- lays eggs at the normal rate. We further compared the life tor phenotype. All of the phenotypes of the dut1-1 mutant span of the ung-1 mutant worms with that of the wildtype are suppressed by the simultaneous inactivation of the UNG. N2 strain. There is no difference between the life span pro- These results suggest that single-strand breaks are accumu- files of the two strains.30) These results suggest the existence lated in the DNA as a result of the UNG-mediated removal of backup enzyme(s). However, as shown in Fig. 3, residual of incorporated dUMP from DNA.33) Interestingly, the uracil excision activity could not be detected in the extract viability of the dut1-1 mutant is strongly impaired by from the ung-1 mutant.30) simultaneous deficiency of the AP endonuclease.34) The anticancer drug 5-fluorouracil affects the level of dUTP URACIL-DNA GLYCOSYLASES IN in the nucleotide pool and increases incorporation of dUMP MAMMALIAN CELLS into DNA. This is one of the mechanisms of 5-fluorouracil cytotoxicity.11) Ung gene disruption in S. cerevisiae has In mammals, there is much more production of uracil protective effects against the lethality of 5-fluorouracil. derived from cytosine deamination than in bacteria. However, These findings suggest a new strategy by which we might it is uncertain whether UNG suppresses spontaneous muta- reduce the adverse side effects of anticancer drugs such as tion in mammals. As shown in Fig. 2, human UNG shows 5-fluorouracil.35) strikingly high identity (40–55%) to UNG glycosylase of Recently, we cloned the ung-1 gene of the nematode C. other organisms.1,3–5,37,38) Furthermore, the exogenous intro- elegans, whose which product (CeUng-1) shares 49% iden- duction of human UNG suppresses the high frequency of tity and 69 % similarity with E. coli UNG.30) The ung-1 gene spontaneous in the E. coli ung mutants.38) of C. elegans is located on chromosome III: 13.69 ± 0.087 In human cells, transgenic Ugi-expressing cells exhibit a cM and has 3 exons, encoding a 282 protein (32 high frequency of spontaneous mutations due to increased KDa). Structural element analysis indicates that this protein, G:C to A:T transitions.39) These facts indicate that human potentially belonging to the UNG family, has eight con- UNG and E. coli UNG possess the same function; that is, served DNA-contacting elements in its sequence:α1~α8 and these remove uracil incorporated into DNA during β1~β4.3,30) These domains are highly conserved among the replication and/or produced by deamination of cytosine in known UNG sequences, including CeUng-1, in the database DNA. The human UNG is gene located at 12q24.1 of chro- (Fig. 2). Purified CeUng-1 removes uracil from both U:G mosome 12 and spans ~13.8 Kb.4,5,40,41) This gene encodes and U:A in double-stranded oligonucleotides. The activity of both mitochondrial UNG1 and nuclear UNG2 isoforms.42,43) CeUng-1 is inhibited by Ugi, a specific inhibitor of the Mitochondrial UNG1 and nuclear UNG2 are the major family-1 UNG,28,29) indicating that CeUng-1 is a member of uracil-DNA glycosylases in human cells.3–5,41–43) These two

Fig. 2. Partial amino acid sequence alignment for C. elegans Ung homolog with H. sapiens, M. musculus and E. coli Ung. Con- served amino acid residues are shown in bold. Portions of the active-site motif A and B of the family 1 Ung are underlined. The highly conserved Gly121-Gln122, Tyr125, Phe136, Apn182, His247, Leu251 residues in CeUng-1 are marked with plus symbols.

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via formation of AP sites.49–53) Enzymatic deamination of cytosine in DNA is catalyzed by AID in activated B cells.51–53) DNA diversification of functional immunoglobulin genes is triggered by AID-mediated deamination of cytosine residues at the immunoglobulin locus, followed by the removal of uracil from U:G lesions by UNG.51–54) Expression of AID in E. coli gives a mutator phenotype that yields G:C to A:T transitions, and such expression in ung mutant cells yields a much higher mutation frequency than the sum of their inde- pendent mutation frequencies.53) Nilsen et al. (2000) reported that UNG knockout mice remained tumor-free and showed no pathological phenotypes for up to 12 months, but beyond 18 months, they showed a higher morbidity and greatly increased incidence of B-cell lymphomas in older mice.46) Fig. 3. Cleavage activity for uracil-containing double-stranded That deficiency in UNG activity affected somatic hypermu- oligonucleotide in extract from C. elegans wild-type and ung-1 tation and class-switch recombination.49) In humans, UNG mutant in the presence or absence of Ugi. The extracts were pre- deficiency was observed in patients with high-IgM ung-1 pared from wild-type N2 (w, lanes 2, 3, 8 and 9) and mutant syndrome. The mutation of phase IA is dominant, as somatic TM2862 (m, lanes 4, 5, 10 and 11) of C. elegans. 32P-labeled U:G hypermutation is deficient in UNG mutant cells.53,54) or U:A (20 fmol) was incubated at 26°C for 5 min with the extract in the presence (lanes 3, 5, 9 and 11) or absence (lanes 2, 4, 8, and Proteins required for the S-S recombination, including γ 10) of 1 unit of Ugi. Lanes 1–6, U:G; lanes 7–12, U:A. Lanes 1 and DNA-PK, ATM, Mre11, Rad50-Nbs1, H2AX, Mdc1 and 7, no enzyme; lanes 6 and 12; 9-mer marker. XRCC4-ligase IV, are important for faithful joining of S regions.55)

UNGs share a common catalytic center but possess different STRUCTURAL ASPECTS OF URACIL-DNA N-terminal amino acids that are derived from two different GLYCOSYLASES transcripts by utilizing promoters PA and PB. Exon 1A of UNG2 is transcribed from promoter PA, and exon 1B of As shown in Fig. 2, CeUng-1 contains certain conserved UNG1 from promoter PB.4,44) Moreover, alternative splicing residues that have counterparts in E. coli and human UNG.30) of the mRNA occurs for each transcript.4,44) The CeUng-1 polypeptide has highly conserved active sites UNG2 co-localizes with PCNA and replication protein A A and B.3,19,30,56–60) Active site A is present between residues (RPA) in replication foci.4,45) UNG2 functions in post- 116~137 in CeUng-1, 58~77 in E. coli UNG and 138~159 replicative removal of uracil incorporated into DNA in in human UNG.19,30,57–59) Active site B is also highly con- mammalian cells.4,45) The specific role of UNG2 in the served between residues 247 and 253 in CeUng-1 (Fig. 1). removal of misincorporated uracil has been demonstrated by The position of a conserved Apn182 is involved in the inhibition of immediate post-replicative removal of and conserved for distinguishing cytosine and uracil, which incorporated uracil in isolated nuclei by neutralizing anti- plays a major role in establishing the high degree of base UNG antibodies,45) and by the slow removal of incorporated specificity displayed by UNG enzyme.3,19,30,56–60) Aromatic uracil in nuclei from UNG–/– mice.46) UNG2 also removes residue, Tyr125, Phe136 and His247 are involved in the uracil from U:G mispairs produced by cytosine deamination stacking interaction with uracil.19,30,58) The sequence in DNA.1,4,5,41–43) It is therefore reasonable to speculate that Gly121-Gln1223,19,56–60) is conserved together with Leu251 the UNG2 dysfunction results in an increased level of uracil in CeUng-1.30) in the DNA of UNG null mice, as described above.46) Although uracil is the main substrate of UNG1 and OTHER ENZYMATIC ACTIVITIES FOR UNG2, these enzymes also remove the oxidized cytosine REMOVAL OF URACIL FROM DNA derivatives isodialuric acid, alloxan and 5-hydroxyuracil.47) Furthermore, Akbari et al. (2007) found that mRNA for UNG is categorized as a family-1 uracil-DNA glycosylase UNG1, but not UNG2, is increased after exposure to H2O2, and its homologous genes are highly conserved in S. indicating regulatory effects of oxidative stress on mitochon- cerevisiae, S. pombe and mammals.3,4,61) Recent studies drial base excision repair.48) It is likely that UNG1 has an revealed that mammalian cells contain at least three important role in the repair of oxidized pyrimidines.47,48) additional families of uracil removing enzymes, classified Recent studies have revealed that UNG has a role in anti- according to the differences in their substrate recognition body diversification related to the activation induced and amino acid sequence (Table 1). They are the MUG cytosine deaminase (AID) and class-switch recombination (mismatch-specific uracil-DNA glycosylase)/TDG (thymine

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Table 1. DNA glycosylase families Family DNA glycosylase E. coli S. cerevisiae S. Pombe C. elegans Mammals Family 1 UNG + + + + + Family 2 MUG/TDG + – + ? + Family 3 SMUG1 – – – – + Family 4 MBD4 – – + – +

DNA glycosylase) family (family-2), the SMUG (single DNA glycosylase activity to remove thymine whose methyl strand- specific monofunctional uracil-DNA glycosylase) group has been oxidized, 5-hydroxymethyluracil and 5- family (family-3) and the MBD4 (methyl-binding domain) formyluracil from DNA.66–69) family (family-4).3–5,19,48,62,63) In C. elegans, UNG removes uracil from both U:G and SMUG1 possesses a uracil-removing activity and func- U:A base pairs in the absence of SMUG.30) However, it is tions as a backup enzyme for UNG. SMUG1 is identified as not likely that C. elegans has any uracil-removing enzymes a new uracil-DNA glycosylase family and found in Xenopus belonging to such other known families, because the C. laevis.64) SMUG1 prefers single-stranded DNA as its sub- elegans database does not reveal any homolog of other fam- strate, but also removes uracil from double-stranded DNA. ily Ung enzymes, including Mug, SMUG1, TDG and UNG-knockout mice (UNG–/–) are fertile and develop GBD4.30) normally to adulthood with no apparent changes in pheno- Two other DNA glycosylases, TDG and MBD4, have type.46,65) Unexpectedly, UNG–/– mice show only slightly been identified in mammals.63,70,71) TDG and MBD4 are elevated spontaneous mutation frequencies. There is an strictly specific for double-stranded DNA, and have very low increase in the mutation frequency in the thymus (1.3-fold) turnover numbers. TDG and MBD4 can repair uracil, thym- and in the spleen (1.4-fold) in the UNG-deficient mice.46,65) ine, and oxidized pyrimidine in methylated CpG sites.63,71–73) In these organs, residual uracil removing activities are the The roles of MBD4 may be limited to repair of mismatched lowest among the organs tested in the mice. However, uracil uracil, thymine and some damaged pyrimidines in double- residues in the genome DNA are increased about 100-fold stranded DNA, particularly in CpG and 5-methyl-CpG con- in the UNG null mice. texts.73) Deficiency of MBD4 in mice enhances mutations at It is reasonable to speculate that, because of the presence CpG sites and alters apoptosis in response to DNA dam- of residual uracil removing activity catalyzed by SMUG1, age.73) MBD4 interacts strongly with MLH.63,71–73) UNG null mice do not exhibit a greatly increased mutation, Uracil in DNA has been thought to occur in different posi- at least overall in the whole genome. Furthermore, UNG and tions, and in addition, the sequence context where it occurs SMUG1 function in different pathways.1,3,47) SMUG1 may be different. It seems likely that the type of uracil- DNA removes uracil in potent mutagenic U:G base pairs produced glycosylase and the mechanism of repair in the downstream by cytosine deamination in DNA.1,3–5) On the other hand, steps will depend on these factors.4) Uracil in the replication UNG has a central function for the removal of uracil from fork may be generated by cytosine deamination, and may be misincorporated dUMP residues, which are not directly removed by any of the four uracil-DNA glycosylases, UNG, mutagenic.45,46) An et al. (2005) reported that stable siRNA- SMUG1, TGD or MBD4. TDG and MBD4 mainly operate mediated knockdown of SMUG1 in mouse embryonic fibro- in the CpG context, while UNG and SMUG1 operate in any blasts has a mutator phenotype.66) An additive 10-fold sequence context.4,73) UNG is a major DNA glycosylase for increase in the frequency of spontaneous G:C to A:T transi- removing uracil from U:G mispairs, as is as SMUG1.4) tions is observed in cells deficient in both SMUG1 and UNG.4,66) These results demonstrate that these enzymes have PERSPECTIVES distinct and non-redundant roles in preventing mutations. Interestingly, cells with mutations in the SMUG1 and UNG Cytosine deamination and the resultant mutations were genes are hypersensitive to ionizing radiation, indicating a thought to be inevitable disasters for cells when people first critical role of the enzymes they encode in the repair of DNA started to study cytosine deamination. It has clearly been damage generated by the oxidation of cytosine. SMUG1 is demonstrated that dUTPase removes dUTP from the nucle- present only in insects and vertebrates,4) and has not been otide pool and there are several uracil-removing enzymes, identified in C. elegans.30) which constitute the of UNG superfamily. Furthermore, the Both UNG2 and SMUG1 are important for the prevention generation of uracil in DNA and its repair mechanism have of mutations caused by cytosine deamination, and their been found to be deeply involved in antigen production functions are non-redundant. Furthermore, SMUG1 has a processes. The following subjects are key for clarifying the

J. Radiat. Res., Vol. 50, No. 1 (2009); http://jrr.jstage.jst.go.jp 24 S.-I. Yonekura et al. mechanisms of the biological effects and repair of cytosine and time-dependent deamination of cytosine bases in UVB- deamination in DNA at the biochemical and molecular induced cyclobutane pyrimidine dimers in vivo. J. Mol. Biol. levels: the binding and regulation of various base excision 284: 297–311. repair enzymes at the damage site; regulation of the expres- 10. Tye, B. K., Chien, J., Lehman, I. R., Duncan, B. K. and sion of uracil DNA glycosylase; physical and functional Warner, H. R. (1978) Uracil incorporation: a source of pulse- labeled DNA fragments in the replication of the Escherichia interactions between UNG and other proteins involved in coli chromosome. Proc. Natl. Acad. Sci. U.S.A. 75: 233–237. base excision repair; functional roles of the different 11. Anderson, S., Heine, T., Sneve, R., Konig, I., Krokan, H. E., glycosylases and coupling to downstream steps in the base Epe, B. and Nilsen, H. (2005) Incorporation of dUMP into excision repair pathway; identification of novel DNA glyc- DNA is a major source of spontaneous DNA damage, while osylases and proteins associated with them. For overviews, excision of uracil is not required for cytotoxicity of fluoropy- the reader should consult previous excellent reviews written rimidines in mouse embryonic fibroblasts. Carcinogenesis 26: by Krokan et al.,1,4) Friedberg et al.,3) Barnes and Lindahl5) 547–555. and Pearl.19) 12. Hagen, L., Peria-Diaz, J., Kavli, B., Otterlei, M., Slupphaug, G. and Krokan, H. E. (2006) Genomic uracil and human disease. Expt. Cell Res. 312: 2666–2672. ACKNOWLEDGEMENTS 13. Lari, S. U., Chen, C. Y., Vertessy, B. G., Morre, J. and Bennett, S. E. (2006) Quantitative determination of uracil We would like to thank the editor-in-chief of the Journal residues in Escherichia coli DNA: Contribution of ung, and of Radiation Research, Dr. Yoshiya Furusawa, for giving us dut genes to uracil avoidance. DNA Repair 5: 1407–1420. the opportunity to write this review article. We also wish to 14. Duncan, B. K., Rockstroh, P. A. and Warner, H. R. (1978) thank Dr. Grigory L. Dianov, Oxford University, for his Escherichia coli K-12 mutants deficient in uracil-DNA glyc- helpful discussion. The studies done in our laboratory were osylase. J. Bacteriol. 134: 1039–1045. financially supported in part by the Global Center of 15. El-Haji, H. H., Zhang, H. and Weiss, B. (1988) Lethality of a Excellence Program [Formation of a Strategic Base for Biodi- dut (deoxyuridine triphosphatase) mutation in Escherichia versity and Evolutionary Research (A06): from Genome to coli. J. Bacterial. 170: 1069–1075. 16. Duncan, B. K. and Weiss, B. (1982) Specific mutator effects Ecosystem] and Grants-in-Aid for Scientific Research of ung (uracil-DNA glycosylase) mutations in Escherichia 19510056 from the Ministry of Education, Culture, Sports coli. J. Bacteriol. 151: 750–755. and Technology, Japan (to QM. Z.). We are grateful to the 17. Eisen, J. A. and Hanawalt, P. (1999) A phylogenomic study Central Research Institute of Electric Power Industry of DNA repair genes, proteins, and processes. Mutat. Res. (Tokyo) for supporting Q-M. Zhang-Akiyama. 435: 171–213. 18. Lindahl, T., Ljungquist, S., Siegert, W., Nyberg, B. and REFERENCES Sperens, B. (1977) DNA N-glycosylases: properties of uracil- DNA glycosylase from Escherichia coli. J. Biol. Chem. 252: 1. Krokan, H. E., Standal, R. and Slupphaug, G. (1997) DNA 3286–3294. glycosylases in the base excision repair of DNA. Biochem. J. 19. Pearl, L. (2000) Structure and function in the uracil-DNA 325: 1–16. glycosylase superfamily. Mutat. Res. 460: 165–181. 2. Bjelland, S. and Seeberg, E. (2003) Mutagenicity, toxicity and 20. Lindahl, T. (1974) An N-glycosylase from Escherichia coli that repair of DNA base damage induced by oxidation. Mutat. Res. releases free uracil from DNA containing deaminated cytosine 531: 37–80. residues. Proc. Natl. Acad. Sci. U.S.A. 71: 3649–3653. 3. Friedberg, E. C., Walker, G. C., Siede, W., Shultz, R. A. and 21. Lindahl, T. (1976) New class of enzymes acting on DNA. Ellenberger, T. (2006) DNA Repair and Mutagenesis, ASM Nature 259: 64–66. Press, Washington. 22. Zhang, Q-M. and Dianov, G. L. (2005) DNA repair fidelity of 4. Krokan, H. E., Drabløs, F. and Slupphaug, G. (2002) Uracil base excision repair pathways in human cell extracts. DNA in DNA-occurrence, consequences and repair. Oncogene 21: Repair 4: 263–270. 8935–8948. 23. Lutsenko, E. and Bhagwat, A. S. (1999) The role of the 5. Barnes, D. E. and Lindahl, T. (2004) Repair and genetic con- Escherichia coli Mug protein in the removal of uracil and sequences of endogenous DNA base damage in mammalian 3,N4-ethenocytosine from DNA. J. Biol. Chem. 274: 31034– cells. Annu. Rev. Genet. 38: 445–476. 31038. 6. Lindahl, T. (1993) Instability and decay of the primary struc- 24. Guo, G., Ding, Y. and Weiss, B. (1997) nfi, the gene for endo- ture of DNA. Nature 362: 709–715. V in Escherichia coli. J. Bacteriol. 179: 310–316. 7. Duncan, B. K. and Miller, J. H. (1980) Mutagenic Deamination 25. Shouten, K. A. and Weiss, B. (1999) Endonuclease V protects of cytosine residues in DNA. Nature 287: 560–561. Escherichia coli against specific mutations caused by nitrous 8. Horsfall, M. J., Borden, A. and Lawrence, C. W. (1997) acid. Mutat. Res. 435: 245–254. Mutagenic properties of the T-C cyclobutane dimer. J. Bacteriol. 26. Guo, G. and Weiss, B. (1998) Endonuclease V (nfi) mutant of 179: 2835–2839. Escherichia coli K-12. J. Bacteriol. 180: 46–51. 9. Tu, Y. Q., Dammann, R. and Pfeifer, G. P. (1998) Sequence 27. Schuster, H. (1960) The reaction of nitrous acid with deoxyri-

J. Radiat. Res., Vol. 50, No. 1 (2009); http://jrr.jstage.jst.go.jp Repair Mechanisms of Cytosine Deamination in DNA. 25

bonucleic acids. Biochem. Biophys. Res. Comm. 2: 320–323. Lindmo, T. and Krokan, H. E. (1998) Nuclear and mitochon- 28. Cone, R., Bonura, T. and Friedberg, E. C. (1980) Inhibitor of drial splice forms of human uracil-DNA glycosylase contain uracil-DNA glycosylase induced by bacteriophage PBS2. a complex nuclear localization signal and a strong classical Purification and preliminary characterization. J. Biol. Chem. mitochondrial localization signal, respectively. Nucleic Acids 255: 10354–10358. Res. 26: 4611–4617. 29. Karran, P., Cone, R. and Friedberg, E. C. (1981) Specificity 43. Krokan, H. E., Otterlei, M., Nilsen, H., Kalvi, B., Skorpen, F., of the bacteriophage PBS2 induced inhibitor of uracil-DNA Anderson, S., Skjelbred, G., Akbari, M., Aas, P. A. and glycosylase. Biochemistry 21: 6092–6096. Slupphaug, G. (2001) Properties and functions of human 30. Nakamura, N., Morinaga, H., Kikuchi, M., Yonekura, S., uracil-DNA glycosylase from the UNG gene. Prog. Nucleic Ishii1, N., Yamamoto, K., Yonei, S. and Zhang, Q-M. (2008) Acids Res. Mol. Biol. 68: 365–386. Cloning and characterization of uracil-DNA glycosylase and 44. Haug, T., Skorpen, F., Aas, P. A., Malm, V., Skjelbred, C. and the biological consequences of the loss of its function in the Krokan, H. E. (1998) Regulation of expression of nuclear and nematode Caenorhabditis elegans. Mutagenesis, in press. mitochondrial forms of human uracil-DNA glycosylase. 31. Impellizzeri, K. J., Anderson, B. and Burgers, P. M. (1991) Nucleic Acids Res. 26: 1449–1457. The spectrum of spontaneous mutations in a Saccharomyces 45. Otterlei, M., Warbrick, E., Nagelhus, T. A., Haug, T., cerevisiae uracil-DNA glycosylase mutant limits the function Slupphaug, G., Akbari, M., Aas, P. A., Steinbekk, K., Bakke, of this enzyme to cytosine deamination repair. J. Bacteriol. O. and Krokan, H. E. (1999) Post-replicative base excision 173: 6807–6810. repair in replication foci. EMBO J. 18: 3834–3844. 32. Chatterjee, A. and Singh, K. K. (2001) Uracil-DNA glycosy- 46. Nilsen, H., Rosewell, I., Robins, P., Skjelbred, C. F., Anderson, lase-deficient yeast exhibits a mitochondria mutator pheno- S., Slupphaug, G., Daly, G., Krokan, H. E., Lindahl, T. and type. Nucleic Acids Res. 29: 4935–4940. Barnes, D. E. (2000) Uracil-DNA glycosylase (UNG)- 33. Gadsden, M. H., McIntosch, E. M., Game, J. C., Wilson, P. J. deficient mice reveal a primary role of the enzyme during and Haynes, R. H. (1993) dUTP pyrophosphatase is an essen- DNA replication. Mol. Cell 5: 1059–1065. tial enzyme in Saccharomyces cerevisiae. EMBO J. 12: 4425– 47. Nilsen, H., Haushalter, K. A., Robins, P., Barnes, D. E., 4431. Verdine, G. L. and Lindahl, T. (2001) Excision of deaminated 34. Guillet, M., van der Kemp, P. A. and Boiteux, S. (2006) cytosine from the vertebrate genome: role of the SMUG1 dUTPase activity is critical to maintain genetic stability in uracil-DNA glycosylase. EMBO J. 20: 4278–4286. Saccharomyces cerevisiae. Nucleic Acids Res. 34: 2056– 48. Akbari, M., Otterlei, M., Pena-Diaz, J. and Krokan, H. E. 2066. (2007) Different organization of base excision repair of uracil 35. Seiple, L., Jaruga, P., Dizdaroglu, M. and Stivers, J. T. (2006) in DNA in nuclei and mitochondria and selective upregulation Linking uracil base excision repair and 5-fluorouracil toxicity of mitochondrial uracil-DNA glycosylase after oxidative in yeast. Nucleic Acids Res. 34: 140–151. stress. Neuroscience 14: 1201–1212. 36. Gengyo-Ando, K. and Mitani, S. (2000) Characterization of 49. Rada, C., Williams, G. T., Nilsen, H., Barnes, D. E., Lindahl, mutations induced by ethyl metahnesulfonate, UV, and T. and Neuberger, M. S. (2002) Immunoglobulin isotype trimethylpsoralen in the nematode Caenorhabditis elegans. switching is inhibited and perturbed in Biochem. Biophys. Res. Comm. 269: 64–69. UNG deficient mice. Curr. Biol. 12: 1748–1755. 37. Olsen, L. C., Aasland, R., Wittwer, C. U., Krokan, H. E. and 50. Imai, K., Slupphaug, G., Lee, W. I., Revy, P., Nonoyama, S., Helland, D. E. (1989) Molecular cloning of human uracil- Catalan, N., Yel, L., Forveille, M., Kavil, B., Krokan, H. E., DNA glycosylase, a highly conserved DNA repair enzyme. Ochs, H. D., Fischer, A. and Durandy, A. (2003) Human EMBO J. 8: 3121–3152. uracil-DNA glycosylase deficiency associated with pro- 38. Olsen, L. C., Aasland, R., Krokan, H. S. and Helland, D. E. foundly impaired immunoglobulin class-switch recombina- (1991) Human uracil DNA glycosylase complements E. coli tion. Nature Immunol. 4: 1023–1028. ung mutants. Nucleic Acids Res. 19: 4473–4478. 51. Longerich, S. and Storb, U. (2005) The contested role of 39. Radany, E. H., Dornfeld, K. J., Sanderson, R. J., Savage, M. uracil-DNA glycosylase in immunoglobulin gene diversifica- K., Majumdar, A., Seidman, M. M. and Mosbaugh, D. W. tion. Trends Genet. 21: 253–256. (2000) Increased spontaneous mutation frequency in human 52. Honjo, T., Muramatsu, M. and Fagarasan, S. (2004) AID: how cells expressing the phage PBS2-encoded inhibitor of uracil does it aid antibody diversity? Immunity 20: 659–668. DNA glycosylase. Mutat. Res. 461: 41–58. 53. Petesen-Mahrt, S. K., Harris, R. S. and Neuberger, M. S. 40. Haug, T., Skorpen, F., Kvaloy, K., Eftedal, I., Lund, H. and (2002) AID mutates E. coli suggesting a DNA deamination Krokan, H. E. (1996) Human uracil-DNA glycosylase gene: mechanism for antibody diversification. Nature 418: 99–103. sequence organization, pattern, and mapping to 54. Di Noia, J. and Neuberger, M. S. (2002) Altering the pathway chromosome 12q23-q24.1. Genomics 36: 408–416. of immunogloburin hypermutation by inhibiting uracil-DNA 41. Slupphaug, G., Eftedal, I., Kavil, B., Bharati, S., Helle, N. M., glycosylase. Nature 419: 43–48. Haug, T., Levine, D. W. and Krokan, H. E. (1995) Properties 55. Stavnezer, J., Guikema, J. E. and Schrader, C. E. (2008) of a recombinant human uracil-DNA glycosylase from the Mechanism and regulation of class switch recombination. UNG gene and evidence that encodes the major uracil-DNA Annu. Rev. Immunol. 26: 261–292. glycosylase. Biochemistry 34: 128–138. 56. Mol, C. D., Arvai, A. S., Slupphaug, G., Kavli, B., Alseph, I., 42. Otterlei, M., Haug, T., Nagelhus, T. A., Slupphaug, G., Krokan, H. E. and Tainer, J. A. (1995) Crystal structure and

J. Radiat. Res., Vol. 50, No. 1 (2009); http://jrr.jstage.jst.go.jp 26 S.-I. Yonekura et al.

mutational analysis of human uracil-DNA glycosylase: 66. An, Q., Robins, P., Lindahl, T. and Barnes, T. E. (2005) C→T structural basis for specificity and catalysis. Cell 80: 869–878. mutagenesis and γ-radiation sensitivity due to deficiency in the 57. Slupphaug, G., Mol, C. D., Kavli, B., Arvai, A. S., Krokan, Smug1 and Ung DNA glycosylases. EMBO J. 24: 2205–2213. H. E. and Tainer, J. A. (1996) A nucleotide-flipping mecha- 67. Boorstein, R. J., Cummings, A., Marenstein, D. R., Chan, M. nism from the structure of human uracil-DNA glycosylase K., Ma. Y., Neubert, T. A., Brown, S. M. and Teebor, G. W. bound to DNA. Nature 384: 87–92. (2001) Definitive identification of mammalian 5- hydroxym- 58. Parikh, S. S., Mol, C. D., Slupphaug, G., Bharati, S., Krokan, ethyluracil DNA N-glycosylase activity as SMUG1. J. Biol. H. E. and Tainer, J. A. (1998) Base excision repair initiation Chem. 276: 41991–41997. revealed by crystal structures and binding kinetics of human 68. Kavil, B., Otterlei, M., Slupphaug, G. and Krokan, H. E. uracil-DNA glycosylase with DNA. EMBO J. 17: 5214–5226. (2007) Uracil in DNAgeneral , but normal intermediate 59. Chung, M. H., Im, E. K., Park, H. Y., Kwon, J. H., Lee, S., in acquired immunity. DNA Repair 6: 505–516. Oh, J. and Hwang, K. C. (2003) A novel uracil-DNA 69. Masaoka, A., Matsubara, M., Hasegawa, R., Tanaka, T., glycosylase family related to the helix-hairpin-helix DNA Kurisu, S., Terato, Ohyama, Y., Karino, N. and Ide, H. (2003) glycosylase superfamily. Nucleic Acids Res. 31: 2045–2055. Mammalian 5-formyluracil-DNA glycosylase. Role of 60. Xiao, G. Y., Tordova, M., Jagadeesh, J., Drohat, A. C., SMUG1 uracil-DNA glycosylase in repair of 5-formyluracil Stivers, J. T. and Gilliland, G. L. (1999) Crystal structure of and other oxidized and deaminated base lesions. Biochemistry Escherichia coli uracil DNA glycosylase and its complexes 42: 5003–5012. with uracil and glycerol: structure and glycosylase mechanism 70. Neddermann, P. and Jiricny, J. (1994) Efficient removal of revisited. Proteins: Struct. Funct. Genet. 35: 13–24. uracil from G.U mispairs by the mismatch-specific thymine 61. Aravind, L. and Koonin, E. V. (2000) The alpha/beta fold DNA glycosylase from HeLa cells. Proc. Natl. Acad. Sci. uracil DNA glycosylases: a common origin with diverse fates. U.S.A. 91: 1642–1646. Genome Biol. 1: 1–8. 71. Hendrick, B. and Bird, A. (1998) Identification and character- 62. Neddermann, P. and Jiricny, J. (1993) The purification of a ization of a family of mammalian methyl CpG binding pro- mismatch-specific thymine-DNA glycosylase from HeLa teins. Mol. Cell. Biol. 18: 6538–6547. cells. J. Biol. Chem. 268: 21218–21224. 72. Hendrick, B., Hardeland, U., Ng, H. N., Jiricny, J. and Bird, 63. Abu, M. and Waters, T. R. (2003) The main role of human A. (1999) The thymine glycosylase MBD4 can bind to the thymine-DNA glycosylase is removal of thymine produced by product of deamination at methylated CpG sites. Nature 401: deamination of 5-methylcytosine and not removal ethenocy- 301–304. tosine. J. Biol. Chem. 278: 8739–8744. 73. Bader, S. A., Walker, M. and Harrison, D. J. (2007) A human 64. Haushalter, K. A., Todd Stukenberg, M. W., Kirschner, M. W. cancer-associated truncation of MBD4 causes dominant and Verdine, G. L. (1999) Identification of a new uracil-DNA negative impairment of DNA repair in colon cancer cells. Br. glycosylase family by expression cloning using synthetic J. Cancer 26: 660–666. inhibitors. Curr. Biol. 9: 174–185. 65. Nilsen, H., Stamp, G., Anderson, S., Hrivnak, G., Krokan, H. Received on July 15, 2008 E., Lindahl, T. and Barnes, D. E. (2003) Gene-targeted mice Revision received on August 26, 2008 lacking the Ung uracil-DNA glycosylase develop B-cell Accepted on August 28, 2008 lymphomas. Oncogene 22: 5381–5386. J-STAGE Advance Publication Date: November 6, 2008

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