Purification and characterization of 3-methyiadenine DNA glycosylase I from<i>Escherichia coli</i>

Bjelland, Svein; Seeberg, Erling

doi:10.1093/nar/15.7.2787

Cited by 82 publications

(99 citation statements)

References 30 publications

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“…ROS-related DNA lesions, including those induced by exogenous H 2 O 2 treatment, are mutagenic, and anaerobic growth conditions reduce their mutagenic effects, such as point mutations and poly(GT) tract instability (13,27,(32)(33)(34)(35). The work presented here demonstrates that reduced oxygen metabolism significantly reduces the rate of spontaneous GCRs in WT strains and tsa1 as well as other mutants and that treatment with H 2 O 2 substantially induces GCRs, indicating that the ROS-induced DNA lesions are an important source of GCRs.…”

Section: Discussionmentioning

confidence: 99%

Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

Ragu

Faye

Iraqui

et al. 2007

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

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The absence of Tsa1, a key peroxiredoxin that functions to scavenge H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations including gross chromosomal rearrangements (GCRs). Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD6 and several key genes involved in DNA double-strand break repair. In the present study we investigated the causes of GCRs and cell death in these mutants. tsa1-associated GCRs were independent of the activity of the translesion DNA polymerases , , and Rev1. Anaerobic growth reduced substantially GCR rates of WT and tsa1 mutants and restored the viability of tsa1 rad6, tsa1 rad51, and tsa1 mre11 double mutants. Anaerobic growth also reduced the GCR rate of rad27, pif1, and rad52 mutants, indicating a role of reactive oxygen species in GCR formation in these mutants. In addition, deletion of TSA1 or H 2O2 treatment of WT cells resulted in increased formation of Rad52 foci, sites of repair of multiple DNA lesions. H 2O2 treatment also induced the GCRs. Our results provide in vivo evidence that oxygen metabolism and reactive oxygen species are important sources of DNA damages that can lead to GCRs and lethal effects in S. cerevisiae.dsDNA break ͉ gross chromosomal rearrangement ͉ translesion DNA synthesis ͉ peroxiredoxin M aintaining genome stability is crucial for cell growth and cell survival. Different genetic disorders, including most human cancers, are associated with different forms of genome instability (1-3). One type of genomic instability observed frequently in many cancers is gross chromosomal rearrangements (GCRs), such as translocations, deletions of chromosome arms, interstitial deletions, inversions, amplifications, chromosome fusions, and aneuploidy. Multiple genes and pathways that suppress GCRs have been characterized by using the yeast Saccharomyces cerevisiae as a model system (4-6). These include genes involved in DNA replication, recombination, and repair, cell-cycle checkpoints that function during DNA replication and repair, and pathways involved in telomere maintenance, chromatin assembly, and detoxification of reactive oxygen species (ROS) (refs. 7 and 8 and references therein). The importance of these pathways for suppressing genome instability is further emphasized by their roles in preventing the development of human cancer (7, 9, 10).Although many pathways that function in the suppression of GCRs have been discovered, little is known about the causes and origin of GCRs. It is generally thought that a major cause of DNA damage that leads to mutations is ROS, which are generated as a normal part of oxygen metabolism but are also produced by ionizing radiation, metabolism of exogenous compounds, and pathological processes such as infection and inflammation (11,12). ROS, such as the superoxide radical, H 2 O 2 , and the hydroxyl radical, can attack almost all cell components and can induce many types of DNA damage, including single-stranded DNA breaks and dsDNA breaks (DSB), base and sugar modificat...

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Section: Discussionmentioning

confidence: 99%

Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

Ragu

Faye

Iraqui

et al. 2007

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

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“…Expression and Purification of B. subtilis and Human AAG ProteinVarious B. subtilis aag constructs were used for expression analysis in BK2118 (pUC19, pT7SCII, and pQE30), ER2566 (pT7SCII), and BL21-RIL codon plus (pT7) at different temperatures (37,22, and 16°C), and the highest expression levels were obtained using BK2118/pUC19 growing at 37°C. 2 liters of cell culture from freshly transformed colonies were grown in LB medium with 100 g/ml ampicillin to A 600 of 1 and induced by 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside for 2 h. Cells were resuspended in 50 mM Tris, pH 7, 500 mM NaCl, and10 mM ␤-mercaptoethanol and sonicated three times for 30 s. (Vibtra Cell Sonicator, Sonics and Materials Inc.).…”

Section: Methodsmentioning

confidence: 99%

“…Alkylbase DNA Glycosylase Assay-10,000 -40,000 dpm (glycosylase assay and HPLC, respectively) of calf thymus DNA alkylated with [ 3 H]methyl-N-nitrosourea (1.5 Ci/mmol; NET-408, PerkinElmer Life Sciences) was incubated with different amounts of cell extracts or purified protein for 30 min at 37°C as described previously (37). The DNA was precipitated with ethanol, and radioactivity in the supernatant was measured in a liquid scintillation counter (Tri-Carb 2900TR, Packard).…”

Section: Methodsmentioning

confidence: 99%

The Bacillus subtilis Counterpart of the Mammalian 3-Methyladenine DNA Glycosylase Has Hypoxanthine and 1,N6-Ethenoadenine as Preferred Substrates

Aamodt

Falnes

Johansen

et al. 2004

Journal of Biological Chemistry

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The AAG family of 3-methyladenine DNA glycosylases was initially thought to be limited to mammalian cells, but genome sequencing efforts have revealed the presence of homologous proteins in certain prokaryotic species as well. Here, we report the first molecular characterization of a functional prokaryotic AAG homologue, i.e. YxlJ, termed bAag, from Bacillus subtilis. The B. subtilis aag gene was expressed in Escherichia coli, and the protein was purified to homogeneity. As expected, B. subtilis Aag was found to be a DNA glycosylase, which releases 3-alkylated purines and hypoxanthine, as well as the cyclic etheno adduct 1,N 6 -ethenoadenine from DNA. However, kinetic analysis showed that bAag removed hypoxanthine much faster than human AAG with a 10-fold higher value for k cat , whereas the rate of excision of 1, N 6 -ethenoadenine was found to be similar. In contrast, it was found that bAag removes 3-methyladenine and 3-methylguanine ϳ10 -20 times more slowly than human AAG, and there was hardly any detectable excision of 7-methylguanine. It thus appears that bAag has a minor role in the repair of DNA alkylation damage and an important role in preventing the mutagenic effects of deaminated purines and cyclic etheno adducts in Bacillus subtilis.Exposure of genomes to a variety of reactive intracellular metabolites and environmental agents results in chemical modifications of the DNA nucleobases, including oxidations, alkylations, and deaminations. Such DNA lesions are primarily repaired through the base excision repair pathway (BER). The first step of BER involves N-glycosylic cleavage of the basesugar bonds by damage-specific DNA glycosylases. The abasic site is cleaved by apurinic/apyrimidinic endonucleases or apurinic/apyrimidinic lyases, and repair is completed through a sequential action of a phosphodiesterase, a DNA polymerase, and a DNA ligase (reviewed in Refs. 1 and 2).Alkylating agents represent one of the most abundant classes of mutagenic and genotoxic agents present in the environment. Repair of alkylation damage is initiated by DNA glycosylases, referred to as 3-methyladenine (3mA) 1 DNA glycosylases, because 3mA is a major substrate for these enzymes.

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“…The Tag protein in E. coli is fairly specific for 3-meA, although it also removes 3-meG with much lower efficiency [134,135] (Figure 2). No other known alkylbase-DNA glycosylase has a similarly narrow substrate specificity.…”

Section: Substrate Specificities Of Dna Glycosylases For Alkylated Basesmentioning

confidence: 99%

DNA glycosylases in the base excision repair of DNA

Krokan

Standal

Slupphaug

1997

Biochemical Journal

771

566

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A wide range of cytotoxic and mutagenic DNA bases are removed by different DNA glycosylases, which initiate the base excision repair pathway. DNA glycosylases cleave the Nglycosylic bond between the target base and deoxyribose, thus releasing a free base and leaving an apurinic\apyrimidinic (AP) site. In addition, several DNA glycosylases are bifunctional, since they also display a lyase activity that cleaves the phosphodiester backbone 3h to the AP site generated by the glycosylase activity. Structural data and sequence comparisons have identified common features among many of the DNA glycosylases. Their active sites have a structure that can only bind extrahelical target bases, as observed in the crystal structure of human uracil-DNA glycosylase in a complex with double-stranded DNA. Nucleotide flipping is apparently actively facilitated by the enzyme. With bacteriophage T4 endonuclease V, a pyrimidine-

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Purification and characterization of 3-methyiadenine DNA glycosylase I fromEscherichia coli

Cited by 82 publications

References 30 publications

Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

The Bacillus subtilis Counterpart of the Mammalian 3-Methyladenine DNA Glycosylase Has Hypoxanthine and 1,N6-Ethenoadenine as Preferred Substrates

DNA glycosylases in the base excision repair of DNA

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