Comparison of Substrate Specificities of <i>Escherichia coli</i> Endonuclease III and Its Mouse Homologue (mNTH1) Using Defined Oligonucleotide Substrates

Asagoshi, Kenjiro; Odawara, Hiroaki; Nakano, Hironobu; Miyano, Takayuki; Terato, Hiroaki; Ohyama, Yoshihiko; Seki, Shuji; Ide, Hiroshi

doi:10.1021/bi000422l

Cited by 54 publications

(44 citation statements)

References 53 publications

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“…The mechanism involving steric exclusion also seems consistent with the observation that Endo III recognizes mFapyG paired with purines more efficiently than that paired with pyrimidines (activity with respect to the paired base: A Ϸ G Ͼ T Ͼ C) (22). Similar to other base lesions (54), steric crash between mFapyG and bulky purines in a helix promotes base flipping into the active-site pocket of Endo III. Thus, the order of f ins (C Ͼ Ͼ T Ϸ A Ͼ G) and f ext (C Ͼ T Ͼ Ͼ A and G) for mFapyG can be interpreted as the combined outcome of hydrogen bonding specific for incoming dCTP and steric exclusion in the polymerase active site.…”

Section: Figsupporting

confidence: 79%

Effects of a Guanine-derived Formamidopyrimidine Lesion on DNA Replication

Asagoshi

Terato

Ohyama

et al. 2002

Journal of Biological Chemistry

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2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/ apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3-5 exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (f ins ‫؍‬ V max /K m for insertion) and extension (f ext ‫؍‬ V max /K m for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative f ins ‫؍‬ 1) > > dTMP (2.4 ؋ 10 ؊4 ) Ϸ dAMP (8.1 ؋ 10 ؊5 ) > dGMP (4.5 ؋ 10 ؊7 )), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative f ext ‫؍‬ 1) > mFapyG:T (4.6 ؋ 10 ؊3 ) > > mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.Reactive oxygen species formed by aerobic metabolism, phagocytic blood cells upon inflammation, ionizing radiation, and photosensitized reactions generate structurally diverse oxidative damage to DNA that stores vital genetic information of cells (1-3). Base lesions thus formed are generally restored by the base excision repair pathway involving multiple enzymes such as N-glycosylase/AP 1 lyase, AP endonuclease, DNA polymerase, and DNA ligase (4, 5). However, if left unrepaired, they result in mutations and/or cell death. It has also been implied that oxidative DNA damage is involved in carcinogenesis and various degenerative diseases (6, 7). 7,8-Dihydro-8-oxoguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) have been identified as major products in the reaction of DNA with reactive oxygen species (Fig. 1A) (1, 2). When formed in DNA, 8-oxoG in a template directs incorporation of non-cognate dAMP as well as cognate dCMP during translesion synthesis by DNA polymerases, thereby inducing GC-to-TA transversions (8, 9). Similarly, when formed in a cellular nucleotide pool, a 2Ј-deoxyribonucleotide form of 8-oxoG can be incorporated opposite adenine as well as cytosine in a DNA template, inducing AT-to-CG transversions (10 -12). Mispair formation between 8-oxoG and adenine leading to mutation involves an unusual syn-conformer of 8-oxoG that can form two hydrogen bonds between O-6 and N-7-H in 8-oxoG and N-6 -H and N-1 in adenine without introducing significan...

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Section: Figsupporting

confidence: 79%

Effects of a Guanine-derived Formamidopyrimidine Lesion on DNA Replication

Asagoshi

Terato

Ohyama

et al. 2002

Journal of Biological Chemistry

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“…Both these observations are in sharp contrast to the in vitro properties of endonuclease III which processes both Tg:G and Tg:A with equal efficiency, yielding the ␤-elimination cleavage product (30). Asagoshi and colleagues (1) concluded that the activity of mNth1 against a Tg:G pair was identical to its activity against a Tg:A pair, but they measured cleavage only at neutral pH. Here we showed that the DNA N-glycosylase activity of mammalian Nth1 is much greater when the Tg residue is opposite an adenine than when opposite a guanine and that an activity which processes Tg:G pairs more efficiently than Tg:A pairs is present in tissue sonicates.…”

Section: Discussionmentioning

confidence: 46%

Targeted Deletion of mNth1 Reveals a Novel DNA Repair Enzyme Activity

Ocampo

Chaung

Marenstein

et al. 2002

Molecular and Cellular Biology

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DNA N-glycosylase/AP (apurinic/apyrimidinic) lyase enzymes of the endonuclease III family (nth in Escherichia coli and Nth1 in mammalian organisms) initiate DNA base excision repair of oxidized ring saturated pyrimidine residues. We generated a null mouse (mNth1 ؊/؊ ) by gene targeting. After almost 2 years, such mice exhibited no overt abnormalities. Tissues of mNth1 ؊/؊ mice contained an enzymatic activity which cleaved DNA at sites of oxidized thymine residues (thymine glycol [Tg]). The activity was greater when Tg was paired with G than with A. This is in contrast to Nth1, which is more active against Tg:A pairs than Tg:G pairs. We suggest that there is a back-up mammalian repair activity which attacks Tg:G pairs with much greater efficiency than Tg:A pairs. The significance of this activity may relate to repair of oxidized 5-methyl cytosine residues (5meCyt). It was shown previously (S. Zuo, R. J. Boorstein, and G. W. Teebor, Nucleic Acids Res. 23:3239-3243, 1995) that both ionizing radiation and chemical oxidation yielded Tg from 5meCyt residues in DNA. Thus, this previously undescribed, and hence novel, back-up enzyme activity may function to repair oxidized 5meCyt residues in DNA while also being sufficient to compensate for the loss of Nth1 in the mutant mice, thereby explaining the noninformative phenotype.Escherichia coli endonuclease III and its eukaryotic homologs initiate the process of DNA base excision repair (BER) of C-5, C-6 ring saturated pyrimidines (31). The endonuclease III-like enzymes are bifunctional enzymes which first effect release of the damaged base from the DNA backbone and then effect strand cleavage at the resulting abasic (apurinic/apyrimidinic [AP]) site via ␤-elimination (19,20,32). Among the modified bases recognized by endonuclease III-like enzymes are 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol [Tg]), cytosine glycol, and cytosine hydrate. Tg is an oxidative product formed in DNA in vitro by reaction with chemical oxidizing agents such as KMnO 4 and OsO 4 as well as via the indirect action of ionizing radiation under aerobic conditions. In cellular DNA, Tg is formed as a product of exposure to aerobic ionizing radiation and other oxidative stresses such as H 2 O 2 in the presence of added Fe 2ϩ . Ionizing irradiation under anaerobic conditions yields the reduced derivative 5,6-dihydrothymine (4, 9, 28). Exposure of cytosine to oxidative stress yields cytosine glycol, which is in equilibrium with its dehydrated form, 5-hydroxycytosine (5ohCyt). 5ohCyt is prone to deamination yielding 5ohUra. In addition to inducing formation of dimeric photoproducts in DNA, UV irradiation also induces hydration of the 5,6 double bond of pyrimidines, primarily forming 5,6 dihydro-6-hydroxy cytosine (cytosine hydrate) (2, 3). Like 5ohCyt, cytosine hydrate is prone to deamination, which yields uracil hydrate (26).Our laboratory first documented the existence of a family of endonuclease III homologs in several species (11) and isolated the cDNA of the human homolog of endonuclease III (hNth...

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“…Accordingly, the pair of 5R cis-trans isomers are abbreviated as 5R-Tg, and the pair of 5S cis-trans isomers as 5S-Tg throughout this paper. 30UR containing a urea residue was prepared by mild alkaline treatment of 30TG5R and 30TG5S (18,41). 25FU, 25HMU, and 25OG containing 5-formyluracil (fU), 5-hydroxymethyluracil (hmU), and 7,8-dihydro-8-oxoguanine (8-oxoG), respectively, were chemically synthesized (42)(43)(44).…”

Section: Methodsmentioning

confidence: 99%

Differential Specificity of Human and Escherichia coli Endonuclease III and VIII Homologues for Oxidative Base Lesions

Katafuchi

Nakano

Masaoka

et al. 2004

Journal of Biological Chemistry

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In human cells, oxidative pyrimidine lesions are restored by the base excision repair pathway initiated by homologues of Endo III (hNTH1) and Endo VIII (hNEIL1 and hNEIL2). In this study we have quantitatively analyzed and compared their activity toward nine oxidative base lesions and an apurinic/apyrimidinic (AP) site using defined oligonucleotide substrates. hNTH1 and hNEIL1 but not hNEIL2 excised the two stereoisomers of thymine glycol (5R-Tg and 5S-Tg), but their isomer specificity was markedly different: the relative activity for 5R-Tg:5S-Tg was 13:1 for hNTH1 and 1.5:1 for hNEIL1. This was also the case for their Escherichia coli homologues: the relative activity for 5R-Tg:5S-Tg was 1:2.5 for Endo III and 3.2:1 for Endo VIII. Among other tested lesions for hNTH1, an AP site was a significantly better substrate than urea, 5-hydroxyuracil (hoU), and guanine-derived formamidopyrimidine (mFapyG), whereas for hNEIL1 these base lesions and an AP site were comparable substrates. In contrast, hNEIL2 recognized an AP site exclusively, and the activity for hoU and mFapyG was marginal. hNEIL1, hNEIL2, and Endo VIII but not hNTH1 and Endo III formed cross-links to oxanine, suggesting conservation of the -fold of the active site of the Endo VIII homologues. The profiles of the excision of the Tg isomers with HeLa and E. coli cell extracts closely resembled those of hNTH1 and Endo III, confirming their major contribution to the repair of Tg isomers in cells. However, detailed analysis of the cellular activity suggests that hNEIL1 has a significant role in the repair of 5S-Tg in human cells.DNA carrying vital genetic information of cells constantly suffers from spontaneous deamination and depurination, alkylation, and oxidation (1-3). These reactions lead to modifications of the DNA backbone and bases, with the latter predominating. The resulting aberrant bases are potentially genotoxic because of the loss or alteration of base pairing information (4), and hence need to be restored by the cellular repair system (2, 3, 5). The major repair mechanism for such damage is the base excision repair (BER) 1 pathway (6), which is conserved from bacteria to humans. In the first step of BER, DNA glycosylases with distinct damage specificities detect the aberrant base in the vast sea of normal bases and remove it from the DNA backbone, leaving an apurinic/apyrimidinic (AP) site. The resulting AP site is further processed and repaired by the subsequent action of AP endonuclease (Endo), DNA polymerase, and DNA ligase through the short patch or long patch BER pathway.The initial search for DNA glycosylases involved in the repair of oxidatively damaged bases in Escherichia coli identified Endo III, Endo VIII, and formamidopyrimidine-DNA glycosylase (7,8). The principal substrates of Endo III and Endo VIII are oxidative pyrimidine lesions. They exhibit redundant damage specificity and catalyze the hydrolysis of the N-glycosidic bond (N-glycosylase activity) and the subsequent incision of an AP site by AP lyase activity via ␤-elimination ...

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Comparison of Substrate Specificities of Escherichia coli Endonuclease III and Its Mouse Homologue (mNTH1) Using Defined Oligonucleotide Substrates

Cited by 54 publications

References 53 publications

Effects of a Guanine-derived Formamidopyrimidine Lesion on DNA Replication

Effects of a Guanine-derived Formamidopyrimidine Lesion on DNA Replication

Targeted Deletion of mNth1 Reveals a Novel DNA Repair Enzyme Activity

Differential Specificity of Human and Escherichia coli Endonuclease III and VIII Homologues for Oxidative Base Lesions

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