Site-specific mutagenesis in vivo by single methylated or deaminated purine bases

Hill-Perkins, M; Jones, Mick D.; Karran, Peter

doi:10.1016/0027-5107(86)90081-3

Cited by 119 publications

(89 citation statements)

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“…Briefly, upon introduction into E. coli cells, the O 6 -alkylguanine adduct located within the unique SmaI restriction site contained in the plasmid is processed by repair pathways and/or undergoes replication. Due to the high miscoding capacity of these adducts (1)(2)(3)(4), unrepaired adducts will yield plasmid progeny containing a majority of G→A transitions at the adduct site, thus inactivating the SmaI restriction site. With single-stranded constructs, the mutant fraction may reach 100% if the adduct is fully miscoding.…”

Section: Resultsmentioning

confidence: 99%

“…Despite their low abundance, O 6 -alkylguanine adducts are responsible for most biological consequences induced by alkylating agents, both in terms of mutagenesis and toxicity. Whereas mutagenesis results from the capacity of these adducts to mispair efficiently with T during replication (1)(2)(3)(4), the way these adducts induce toxicity is more cumbersome. Indeed, cytotoxicity involves the mismatch repair (MMR) system that recognizes the mutagenic O 6 -methylguanine: T (mG:T) replication intermediates.…”

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confidence: 99%

“…contributed equally to this work. 3 To whom correspondence should be addressed. E-mail: fuchs@ifr88.cnrs-mrs.fr.…”

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confidence: 99%

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Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O ⁶ -alkylguanine adducts in Escherichia coli

Mazón

Philippin

Cadet

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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O 6 -alkylG adducts are highly mutagenic due to their capacity to efficiently form O 6 -alkylG:T mispairs during replication, thus triggering G→A transitions. Mutagenesis is largely prevented by repair strategies such as reversal by alkyltransferases or excision by nucleotide excision repair (NER). Moreover, methyl-directed mismatch repair (MMR) is known to trigger sensitivity to methylating agents via a mechanism that involves recognition by MutS of the O 6 -mG:T replication intermediates. We wanted to investigate the mechanism by which MMR controls the genotoxicity of environmentally relevant O 6 -alkylG adducts formed by ethylene oxide and propylene oxide. Recently, the alkyltransferase-like gene ybaZ (eATL) was shown to enhance repair of these slightly larger O 6 -alkylG adducts by NER. We analyzed the toxicity and mutagenesis induced by these O 6 -alkylG adducts using single-adducted plasmid probes. We show that the eATL gene product prevents MMR-mediated attack of the O 6 -alkylG:T replication intermediate for the larger alkyl groups but not for methyl. In vivo data are compatible with the occurrence of repeated cycles of MMR attack of the O 6 -alkylG:T intermediate. In addition, in vitro, the eATL protein efficiently prevents binding of MutS to the O 6 -alkylG:T mispairs formed by the larger alkyl groups but not by methyl. In conclusion, eATL not only enhances the efficiency of repair of these larger adducts by NER, it also shields these adducts from MMR-mediated toxicity.futile mismatch repair cycling | interference between repair pathways | nucleotide excision repair | ybaZ gene

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O ⁶ -alkylguanine adducts in Escherichia coli

Mazón

Philippin

Cadet

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…With respect to mutagenesis, while there is some controversy about the ability of hypoxanthine to induce mutations in E. coli (4,44), dI, dX, and dU are all established mutagens in higher organisms. So it is reasonable to assume that genetic polymorphisms that raise the steady-state levels of dX and dI in DNA in humans would exacerbate the toxicity associated with the nitrosative stress of chronic inflammation, with further increases in dX and dI exceeding the cell's repair capacity and contributing to mutations and cancer risk.…”

Section: Discussionmentioning

confidence: 99%

Defects in purine nucleotide metabolism lead to substantial incorporation of xanthine and hypoxanthine into DNA and RNA

Pang

McFaline

Burgis

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

111

110

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Deamination of nucleobases in DNA and RNA results in the formation of xanthine (X), hypoxanthine (I), oxanine, and uracil, all of which are miscoding and mutagenic in DNA and can interfere with RNA editing and function. Among many forms of nucleic acid damage, deamination arises from several unrelated mechanisms, including hydrolysis, nitrosative chemistry, and deaminase enzymes. Here we present a fourth mechanism contributing to the burden of nucleobase deamination: incorporation of hypoxanthine and xanthine into DNA and RNA caused by defects in purine nucleotide metabolism. Using Escherichia coli and Saccharomyces cerevisiae with defined mutations in purine metabolism in conjunction with analytical methods for quantifying deaminated nucleobases in DNA and RNA, we observed large increases (up to 600-fold) in hypoxanthine in both DNA and RNA in cells unable to convert IMP to XMP or AMP (IMP dehydrogenase, guaB; adenylosuccinate synthetase, purA, and ADE12), and unable to remove dITP/ITP and dXTP/XTP from the nucleotide pool (dITP/XTP pyrophosphohydrolase, rdgB and HAM1). Conversely, modest changes in xanthine levels were observed in RNA (but not DNA) from E. coli lacking purA and rdgB and the enzyme converting XMP to GMP (GMP synthetase, guaA). These observations suggest that disturbances in purine metabolism caused by known genetic polymorphisms could increase the burden of mutagenic deaminated nucleobases in DNA and interfere with gene expression and RNA function, a situation possibly exacerbated by the nitrosative stress of concurrent inflammation. The results also suggest a mechanistic basis for the pathophysiology of human inborn errors of purine nucleotide metabolism.DNA and RNA damage | mass spectrometry | nucleobase deamination | purine metabolism | DNA repair T he chemical modification of nucleobases in DNA and RNA can arise from both physiological and adventitious mechanisms at all stages of nucleic acid metabolism. This is particularly true for deaminated versions of the nucleobases. As shown in Fig. 1 for purines, nucleobase deamination in DNA and RNA leads to the formation of 2′-deoxy-and ribonucleoside forms of hypoxanthine (2′-deoxyinosine, dI; inosine, Ino) from adenine, xanthine (2′-deoxyxanthosine, dX; xanthosine, Xao) and oxanine (2′-deoxyoxanosine, dO; oxanosine, Oxo) from guanine, and uracil (2′-deoxyuridine, dU; uridine, Urd) from cytosine (1). All of these products are miscoding and mutagenic in DNA (2-4) and can interfere with RNA editing (5) and the function of noncoding RNAs (6).There are three recognized mechanisms that contribute to nucleobase deamination in DNA and RNA, the simplest of which is hydrolysis (7). A second source of nucleobase deamination is associated with the nitrosative stress caused by increases in nitric oxide-derived nitrous anhydride during inflammation (1). A third mechanism is associated with deaminase enzymes acting on RNA and DNA, with activation-induced cytidine deaminase converting cytidine to uridine during immunoglobulin diversification in B lymphocytes an...

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“…The net biological impact of a lesion depends on the balance between the ability of the cell to repair the lesion and the mutagenic and toxic properties of the lesion. One particularly critical type of DNA alkylation damage is O 6 -methylguanine (O 6 mG), which has been shown to be both mutagenic [1][2][3][4] and, when cells cannot distinguish nascent from parental DNA strands, toxic [5][6][7]. Extensive in vivo mutagenesis studies done in our laboratory and others established that O 6 mG codes as adenine during replication in E. coli nearly 100% of the time [1,8].…”

Section: Introductionmentioning

confidence: 99%

Mismatch repair proteins collaborate with methyltransferases in the repair of O6-methylguanine

et al. 2008

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DNA repair is essential for combatting the adverse effects of damage to the genome. One example of base damage is O 6 -methylguanine (O 6 mG), which stably pairs with thymine during replication and thereby creates a promutagenic O 6 mG:T mismatch. This mismatch has also been linked with cellular toxicity. Therefore, in the absence of repair, O 6 mG:T mismatches can lead to cell death or result in G:C→A:T transition mutations upon the next round of replication. Cysteine thiolate residues on the Ada and Ogt methyltransferase (MTase) proteins directly reverse the O 6 mG base damage to yield guanine. When a cytosine is opposite the lesion, MTase repair restores a normal G:C pairing. However, if replication past the lesion has produced an O 6 mG:T mismatch, MTase conversion to a G:T mispair must still undergo correction to avoid mutation. Two mismatch repair pathways in E. coli that convert G:T mispairs to native G:C pairings are methyl-directed mismatch repair (MMR) and very short patch repair (VSPR). This work examined the possible roles that proteins in these pathways play in coordination with the canonical MTase repair of O 6 mG:T mismatches. The possibility of this repair network was analyzed by probing the efficiency of MTase repair of a single O 6 mG residue in cells deficient in individual mismatch repair proteins (Dam, MutH, MutS, MutL, or Vsr). We found that MTase repair in cells deficient in Dam or MutH showed wild-type levels of MTase repair. In contrast, cells lacking any of the VSPR proteins MutS, MutL, or Vsr showed a decrease in repair of O 6 mG by the Ada and Ogt MTases. Evidence is presented that the VSPR pathway positively influences MTase repair of O 6 mG:T mismatches, and assists the efficiency of restoring these mismatches to native G:C base pairs.

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Site-specific mutagenesis in vivo by single methylated or deaminated purine bases

Cited by 119 publications

References 23 publications

Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O ⁶ -alkylguanine adducts in Escherichia coli

Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O ⁶ -alkylguanine adducts in Escherichia coli

Defects in purine nucleotide metabolism lead to substantial incorporation of xanthine and hypoxanthine into DNA and RNA

Mismatch repair proteins collaborate with methyltransferases in the repair of O6-methylguanine

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Site-specific mutagenesis in vivo by single methylated or deaminated purine bases

Cited by 119 publications

References 23 publications

Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O 6 -alkylguanine adducts in Escherichia coli

Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O 6 -alkylguanine adducts in Escherichia coli

Defects in purine nucleotide metabolism lead to substantial incorporation of xanthine and hypoxanthine into DNA and RNA

Mismatch repair proteins collaborate with methyltransferases in the repair of O6-methylguanine

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Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O ⁶ -alkylguanine adducts in Escherichia coli

Alkyltransferase-like protein (eATL) prevents mismatch repair-mediated toxicity induced by O ⁶ -alkylguanine adducts in Escherichia coli