The Role of the Escherichia coli Mug Protein in the Removal of Uracil and 3,N 4-Ethenocytosine from DNA

Lutsenko, Eugene A.; Bhagwat, Ashok S.

doi:10.1074/jbc.274.43.31034

Cited by 59 publications

(63 citation statements)

References 20 publications

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“…Lloyd (Oregon Health Sciences University). [41] was a gift from A. Bhagwat (Wayne State University). We believe that the mug gene is more correctly referred to as dug, for double-strand uracil-DNA glycosylase as this activity was originally named when first detected by Gallinari and Jiricny [42], and later characterized by Sung [43].…”

Section: Methodsmentioning

confidence: 99%

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

et al. 2006

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The steady-state levels of uracil residues in DNA extracted from strains of Escherichia coli were measured and the influence of defects in the genes for uracil-DNA glycosylase (ung), doublestrand uracil-DNA glycosylase (dug), and dUTP pyrophosphatase (dut) on uracil accumulation was determined. A sensitive method, called the Ung-ARP assay, was developed that utilized E. coli Ung, T4pdg, and the Aldehyde Reactive Probe reagent to label a basic sites resulting from uracil excision with biotin. The limit of detection was one uracil residue per million DNA nucleotides (U/10 6 nt). Uracil levels in the genomic DNA of E. coli JM105 (ung + dug + ) were at the limit of detection, as were those of an isogenic dug mutant, regardless of growth phase. Inactivation of ung in JM105 resulted in 31 ± 2.6 U/10 6 nt during early log growth and 19 ± 1.7 U/ 10 6 nt in saturated phase. An ung dug double mutant (CY11) accumulated 33 ± 2.9 U/10 6 nt and 23 ± 1.8 U/10 6 nt during early log and saturated phase growth, respectively. When cultures of CY11 were supplemented with 20 ng/ml of 5-fluoro-2′-deoxyuridine, uracil levels in early log phase growth DNA rose to 125 ± 1.7 U/10 6 nt. Deoxyuridine supplementation reduced the amount of uracil in CY11 DNA, but uridine did not. Levels of uracil in DNA extracted from CJ236 (dut-1 ung-1) were determined to be 3000-8000 U/10 6 nt as measured by the Ung-ARP assay, twodimensional thin-layer chromatography of metabolically-labeled 32 P DNA, and LC/MS of uracil and thymine deoxynucleosides. DNA sequencing revealed that the sole molecular defect in the CJ236 dUTP pyrophosphatase gene was a C→T transition mutation that resulted in a Thr24Ile amino acid change.

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Section: Methodsmentioning

confidence: 99%

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

et al. 2006

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“…Because G ⅐T mismatches produced by the deamination of 5-methylcytosine occur exclusively in the sequence context CpG ⅐T, the main role of TDG in cells must therefore be to remove deaminated 5-methylcytosine from CpG sites. In E. coli the mismatch-specific uracil-DNA glycosylase, which is a homologue of TDG, appears to be the only glycosylase that removes ethenocytosine efficiently (35). In humans, besides TDG there are two other enzymes known to remove ethenocytosine.…”

Section: Discussionmentioning

confidence: 99%

The Main Role of Human Thymine-DNA Glycosylase Is Removal of Thymine Produced by Deamination of 5-Methylcytosine and Not Removal of Ethenocytosine

Abu

Waters

2003

Journal of Biological Chemistry

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؊1 . The next best substrates were DNA duplexes containing TpG ⅐ ⑀ C, GpG ⅐ ⑀ C, and CpG⅐T. These had specificity constants 45-130 times smaller than CpG ⅐ ⑀ C-DNA. The worst substrates were DNA duplexes containing ApG ⅐ ⑀ C and TpG ⅐T, which had specificity constants, respectively, 1,600 and 7,400 times lower than CpG ⅐ ⑀ C-DNA. DNA containing ethenocytosine was bound much more tightly than DNA containing a G ⅐T mismatch. This is probably because thymine-DNA glycosylase can flip out ethenocytosine from a G ⅐ ⑀ C base pair more easily than it can flip out thymine from a G ⅐T mismatch. Because thymine-DNA glycosylase has a larger specificity constant for the removal of ethenocytosine, it has been suggested its primary purpose is to deal with ethenocytosine. However, these results showing that thymine-DNA glycosylase has a strong sequence preference for CpG sites in the excision of both thymine and ethenocytosine suggest that the main role of thymine-DNA glycosylase in vivo is the removal of thymine produced by deamination of 5-methylcytosine at CpG sites.It has been known for nearly 30 years that exposure to vinyl chloride can cause cancer in humans (1). Vinyl chloride is metabolized by cytochrome P450 2E1 to form chloroethylene oxide (2) which can rearrange spontaneously to give chloroacetaldehyde (3). Both these metabolites react in vitro with DNA to form ethenoadducts of adenine, guanine, and cytosine (Fig. 1). Three of these four possible ethenobases have been detected in animals exposed to vinyl chloride (reviewed in Ref. 4). Ethenobases have also been found in the DNA of rats and humans not exposed to vinyl chloride. These are probably formed endogenously by the reaction of lipid peroxidation products with DNA (5). The ethenobases cause mutations by misincorporating during DNA replication, and there is evidence that these mutations are responsible for the carcinogenicity of vinyl chloride and related chemicals (6).Extracts from human cells remove all four ethenoadducts from DNA (7). Because they are released from the DNA as the ethenobases, it is likely that they are repaired by the base excision repair pathway. The base excision repair pathway (reviewed in Refs. 8 and 9) involves initial removal of the damaged base by a DNA glycosylase. In the short-patch repair pathway the resultant abasic site is cut by an apurinic endonuclease, probably human apurinic endonuclease 1 (APEX 1 ; also known as HAP1, APE1, or Ref-1). The single nucleotide gap is filled by DNA polymerase ␤ which also removes the abasic sugar-phosphate. Finally, the phosphate backbone is restored by a DNA ligase. Thymine-DNA glycosylase (TDG) is the enzyme believed to repair G ⅐T mismatches arising from spontaneous deamination of 5-methylcytosine (10). Support for this comes from the observation that TDG excises thymine from G ⅐T mismatches at sites of cytosine methylation (i.e. CpG) much more efficiently than from other DNA sequences (11)(12)(13)(14). Recently, two groups (15, 16) independently found that TDG can also remove ethenocytosine f...

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“…E. coli Ndk, similar to its human counterparts, is capable of cleaving DNA (29), an activity that was described by Postel and Abramczyk (28) as related to uracil processing. Prior to that, all of the uracil-DNA glycosylase (UDG) activity in extracts of E. coli has been attributed to either Ung, the major uracil-DNA glycosylase, or to Mug, an auxiliary enzyme (30,31). Furthermore, any association, intrinsic or extrinsic, of a uracil-DNA glycosylase activity with Ndk was ruled out by Bennett et al (32) and Kumar et al (33) on the bases that Ndk purified from Ungdeficient mutant bacteria did not possess UDG activity and that there was no evidence of a physical interaction between Ndk and Ung (32,33).…”

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confidence: 99%

Molecular and Functional Interactions between Escherichia coli Nucleoside-diphosphate Kinase and the Uracil-DNA Glycosylase Ung

Goswami¹,

Yoon

Abramczyk

et al. 2006

Journal of Biological Chemistry

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Escherichia coli nucleoside-diphosphate kinase (Ndk) catalyzes nucleoside triphosphate synthesis and maintains intracellular triphosphate pools. Mutants of E. coli lacking Ndk exhibit normal growth rates but show a mutator phenotype that cannot be entirely attributed to the absence of Ndk catalytic activity or to an imbalance in cellular triphosphates. It has been suggested previously that Ndk, similar to its human counterparts, possesses nuclease and DNA repair activities, including the excision of uracil from DNA, an activity normally associated with the Ung and Mug uracil-DNA glycosylases (UDGs) in E. coli. Here we have demonstrated that recombinant Ndk purified from wild-type E. coli contains significant UDG activity that is not intrinsic, but rather, is a consequence of a direct physical and functional interaction between Ung and Ndk, although a residual amount of intrinsic UDG activity exists as well. Co-purification of Ung and Ndk through multicolumn low pressure and nickel-nitrilotriacetic acid affinity chromatography suggests that the interaction occurs in a cellular context, as was also suggested by co-immunoprecipitation of endogenous Ung and Ndk from cellular extracts. Glutathione S-transferase pulldown and far Western analyses demonstrate that the interaction also occurs at the level of purified protein, suggesting that it is specific and direct. Moreover, significant augmentation of Ung catalytic activity by Ndk was observed, suggesting that the interaction between the two enzymes is functionally relevant. These findings represent the first example of Ung interacting with another E. coli protein and also lend support to the recently discovered role of nucleoside-diphosphate kinases as regulatory components of multiprotein complexes. Escherichia coli nucleoside-diphosphate (NDP)3 kinase (NDK, EC 2.7.4.6) belongs to a large family of highly conserved oligomeric phosphate transfer enzymes consisting of 4 -6 identically folded subunits of 16 -20 kDa, each containing an active site. NDP kinases catalyze the reversible transfer of ␥-phosphates between nucleoside di-and triphosphates at very high efficiencies through an evolutionarily conserved active site histidine residue (1-3). E. coli NDP kinase is encoded by a single gene, ndk (4), whereas the genetically distinct forms of human NDP kinases are encoded by multiple genes termed NM23-H1 through H8 (5). The name NM (nonmetastatic)23 was initially accorded to the matriarch of the family, NM23-H1, on the basis of its reported action as a tissue-specific metastasis inhibitor (6). Moreover, there is evidence that NM23/NDK proteins promote tumor formation and play various roles in normal development and cellular proliferation (7,8).NM23/NDP kinases are also multifunctional in vitro. In addition to the phosphoryl transfer reactions, they can catalyze various types of DNA cleavage (9 -12) and activate transcription (13-15). The involvement of NM23 in DNA repair was initially proposed on the basis of a covalent, lysine-mediated mechanism by which NM23-H2/NDK-B ...

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The Role of the Escherichia coli Mug Protein in the Removal of Uracil and 3,N 4-Ethenocytosine from DNA

Cited by 59 publications

References 20 publications

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

The Main Role of Human Thymine-DNA Glycosylase Is Removal of Thymine Produced by Deamination of 5-Methylcytosine and Not Removal of Ethenocytosine

Molecular and Functional Interactions between Escherichia coli Nucleoside-diphosphate Kinase and the Uracil-DNA Glycosylase Ung

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