The Pyrimidine Ring-opened Derivative of 1,N 6-Ethenoadenine Is Excised from DNA by theEscherichia coli Fpg and Nth Proteins

Speina, Elz ̇bieta; Cieśla, Jarosław M.; Wójcik, Jacek; Bajek, Monika; Kuśmierek, Jarosław T.

doi:10.1074/jbc.m100998200

Cited by 26 publications

(21 citation statements)

References 32 publications

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“…Since we and others have previously shown that ethenoadenine decomposes slowly into derivatives, which are not recognized by ANPG glycosylase, which excises ethenoA [62,63], and other ethenoadducts are also unstable, we have taken this into consideration during calculation of the results. In each experiment the negative control reaction without AlkB protein was performed, and percent of “repair” was calculated also for this negative control reaction.…”

Section: Methodsmentioning

confidence: 99%

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

et al. 2015

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AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno (ε)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ε-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ε-adducts, 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC) and 1,N2-ethenoguanine (1,N2-εG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ε-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed εA and εC from ds and ssDNA but were inactive toward 1,N2-εG. SC-1A repaired only εA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ε-adducts in dsDNA, while only εA and εC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only εC in ssDNA Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N2-εG and that ALKBH3 removes only εC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins.

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Section: Methodsmentioning

confidence: 99%

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

et al. 2015

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“…Aliquots were withdrawn at various times and quenched with 2 volumes of 0.3 M sodium hydroxide to obtain 0.2 M final concentration. The quenched samples were stored at 4 °C until further processed to prevent base-catalyzed ring-opening and subsequent depurination of the εA lesions (32). Samples were heated at 70 °C for 15 minutes to quantitatively cleave abasic sites.…”

Section: Methodsmentioning

confidence: 99%

Human Alkyladenine DNA Glycosylase Employs a Processive Search for DNA Damage

2008

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DNA repair proteins conduct a genome-wide search to detect and repair sites of DNA damage wherever they occur. Human alkyladenine DNA glycosylase (AAG) is responsible for recognizing a variety of base lesions, including alkylated and deaminated purines, and initiating their repair via the base excision repair pathway. We have investigated the mechanism by which AAG locates sites of damage using an oligonucleotide substrate containing two sites of DNA damage. This substrate was designed so that AAG randomly binds to either of the two lesions. AAG-catalyzed base excision creates a repair intermediate and the subsequent partitioning between dissociation and diffusion to the second site can be quantified from the rates of formation of the different products. Our results demonstrate that AAG has the ability to slide for short distances along DNA at physiological salt concentrations. The processivity of AAG decreases with increasing ionic strength to become fully distributive at high ionic strength, suggesting that electrostatic interactions between the negatively charged DNA and the positively charged DNA binding surface are important for nonspecific DNA binding. Although the amino terminus of the protein is dispensable for glycosylase activity at a single site, we find that deletion of the amino terminal 80 amino acids significantly decreases the processivity of AAG. These observations support the idea that diffusion on undamaged DNA contributes to the search for sites of DNA damage.Although DNA is remarkably stable, it is nevertheless susceptible to spontaneous damage via reactions with cellular metabolites and environmental mutagens. Chemical reactions that alter the structure of the nucleobases within DNA are most commonly recognized and repaired by the base excision repair (BER) 1 pathway. Some base lesions can block DNA replication and transcription with cytotoxic effects, and many more alter the base pairing properties so that replication leads to mispairing and mutation. The base excision repair pathway is initiated by a DNA repair glycosylase that must locate the site of damage within the genome. Once a damaged base is located, the glycosylase flips out the damaged nucleotide and catalyzes the hydrolysis of the N-glycosidic bond to release the lesioned base. The subsequent actions of an endonuclease, abasic site lyase, DNA polymerase and DNA ligase are required to complete the repair pathway.It is estimated that ∼10,000 base lesions are formed in a typical human cell every day and that the vast majority of these are correctly repaired by BER or other DNA repair pathways (4). On the one hand, this is a large number of potential mutagenic events that must be corrected. On the other hand, these lesions are very rare considering the size of the human diploid genome † This work was supported by a grant from the NIH to P.O. (CA122254).* Address correspondence to P.O. at the Department of Biological Chemistry, University of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0606. E-mai...

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“…UGI (uracil-DNA glycosylase inhibitor), a viral protein, has been shown to inhibit uracil-DNA glycosylase, which excises uracils in DNA generated from replication misincorporations (34). Speina et al (35) screened a battery of compounds including various base analogs and tryptophan pyrolysate (Trp-P-1), a mutagenic DNA intercalator heterocyclic amine found in cooked food, which targets a number of E. coli and human DNA glycosylases. The 2-thioxanthine was effective for E. coli Fpg protein and was shown to inhibit its excision activity whereas Trp-P-1 inhibits multiple DNA glycosylases such as AlkA, TagA, MPG, Nth, and Fpg by altering the DNA secondary structures (35).…”

Section: Discussionmentioning

confidence: 99%

Magnesium, Essential for Base Excision Repair Enzymes, Inhibits Substrate Binding of N-Methylpurine-DNA Glycosylase

Adhikari

Toretsky

Yuan

et al. 2006

Journal of Biological Chemistry

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is an absolute requirement for the downstream activities of the major base excision repair enzymes, it may act as a regulator for the base excision repair pathway for efficient and balanced repair of damaged bases, which are often less toxic and/or mutagenic than their subsequent repair product intermediates.Cellular DNA is continuously exposed to endogenous or exogenous chemical or physical agents that induce DNA lesions. DNA base damage threatens genomic stability and cellular viability. Multiple DNA repair pathways exist in all organisms, from bacteria to humans, to preserve the integrity of the genome (1). If not repaired, damaged bases could be mutagenic (2) and/or cause cell death by blocking DNA replication (3).In all organisms, repair of DNA-containing small adducts, as well as altered and abnormal bases, occurs primarily via the base excision repair (BER) 2 pathway, beginning with cleavage of the base by a DNA glycosylase (1, 2). Mechanistically, DNA glycosylases are categorized into two classes: mono-and bifunctional DNA glycosylases. Monofunctional DNA glycosylases, such as N-methylpurine-DNA glycosylase (MPG) and uracil-DNA glycosylase, use an activated water molecule as a nucleophile to generate an apurinic or apyrimidinic (AP) site in DNA. Bifunctional DNA glycosylases/AP lyases, such as NTH1 and OGG1, use an activated amino group (Lys) or imino group (Pro) as the nucleophile to create a Schiff base intermediate that coordinates base removal and subsequent strand incision (AP lyase) 3Ј to the AP site (4, 5). The mammalian MPG is known to excise at least 17 structurally diverse modified bases from DNA (6). These lesions include 3-alkylpurines, 7-alkylguanine, 1,N 6 -ethenoadenine (⑀A), N 2 ,3-ethenoguanine, and hypoxanthine (Hx), all of which are purine derivatives (7-12). Moreover, the base alterations are located in both the major and minor grooves of duplex DNA. Its orthologs in Escherichia coli (AlkA) and yeast (MAG) have an overlapping although not identical substrate range. Nonetheless mammalian MPG and E. coli AlkA do not share significant sequence similarity or structural homology (13,14), despite this functional similarity and the fact that 3-methyladenine is a preferred substrate for both. MPG excises ⑀A and Hx more efficiently than AlkA and MAG (11), but unlike AlkA, it cannot excise O 2 -alkylpyrimidines (15, 16) and oxidized bases such as 5-formyluracil and 5-hydroxymethyluracil (17) (22); however, the reduction was more pronounced for the AP-lyase activity. The Schiff base formation between hOGG1-and 8-oxoG-containing DNA was abrogated in the presence of Mg 2ϩ . These results suggest that hOGG1 operates mainly as a monofunctional glycosylase under physiologic concentrations of Mg 2ϩ (22). There

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The Pyrimidine Ring-opened Derivative of 1,N 6-Ethenoadenine Is Excised from DNA by theEscherichia coli Fpg and Nth Proteins

Cited by 26 publications

References 32 publications

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

Human Alkyladenine DNA Glycosylase Employs a Processive Search for DNA Damage

Magnesium, Essential for Base Excision Repair Enzymes, Inhibits Substrate Binding of N-Methylpurine-DNA Glycosylase

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