DNA N-glycosidases: properties of uracil-DNA glycosidase from Escherichia coli.

Lindahl, Tomas; Ljungquist, Siv; Siegert, W.; Nyberg, Barbro; Sperens, B

doi:10.1016/s0021-9258(17)40386-3

Cited by 504 publications

(160 citation statements)

References 49 publications

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“…The protein did not lose activity when pre-incubated without substrate at 75°C for 20 minutes. The UDG of E. coli has been shown to lose 80% of its activity when incubated at 50°C for 5 minutes [13]. The T. maritima enzyme also showed activity at 37°C.…”

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confidence: 99%

Thermostable uracil-DNA glycosylase from Thermotoga maritima a member of a novel class of DNA repair enzymes

Sandigursky

Franklin

1999

Current Biology

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Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in eukaryotes and prokaryotes [1][2][3]. This enzyme removes uracil bases that are present in DNA as a result of either deamination of cytosine or misincorporation of dUMP instead of dTMP [4] [5], and it is the primary activity in the DNA base excision repair pathway. Although UDG activities have been shown to be present in several thermophiles [6][7][8], no sequences have been found that are complementary to the Escherichia coli ung gene, which encodes UDG [9]. Here, we describe a UDG from the thermophile Thermotoga maritima. The T. maritima UDG gene has a low level of homology to the E. coli G-T/U mismatch-specific DNA glycosylase gene (mug). The expressed protein is capable of removing uracil from DNA containing either a U-A or a U-G base pair and is heat-stable up to 75 degrees C. The enzyme is also active on single-stranded DNA containing uracil. Analogous genes appear to be present in several prokaryotic organisms, including thermophilic and mesophilic eubacteria as well as archaebacteria, the human-disease pathogens Treponema palladium and Rickettsia prowazekii, and the extremely radioresistant organism Deinococcus radiodurans. These findings suggest that the T. maritima UDG is a member of a new class of DNA repair enzymes.

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confidence: 99%

Thermostable uracil-DNA glycosylase from Thermotoga maritima a member of a novel class of DNA repair enzymes

Sandigursky

Franklin

1999

Current Biology

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“…The results yielded ∼30× coverage of each genome, which is sufficient to detect single-nucleotide polymorphisms (SNPs) with a high confidence score ( 2 ). An analysis of the deep DNA-seq revealed that the ung gene, encoding the enzyme responsible for removing uracil bases from DNA ( 4 , 5 ), was specifically mutated in all the 015-resistant clones ( SI Appendix , Table S1 ). The mutations were then confirmed using Sanger sequencing.…”

Section: Resultsmentioning

confidence: 99%

“…The 015 gene product forms a complex with the host Ung (2) and thus localizes to newly formed AP sites (3). 015 then attacks the AP site (4) and forms a nick in the chromosomal DNA (5), which leads to DNA replication arrest and ultimately to cell death (6).…”

Section: Methodsmentioning

confidence: 99%

“…It excises uracils misincorporated into the DNA as a result of erroneous use of the deoxyuridine 5′-triphosphate (dUTP) nucleotide by DNA polymerase or due to deamination of cytosine ( 4 ). Release of the uracil from the DNA results in a potentially cytotoxic abasic site (known as an apurinic/apyrimidinic site; henceforth referred to as an AP site) because it blocks DNA synthesis ( 5 ). AP endonuclease activities such as those associated with exonuclease III, endonuclease III, and endonuclease IV can then cleave the DNA at the resulting AP sites, resulting in a nicked DNA strand ( 6 ).…”

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confidence: 99%

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A phage mechanism for selective nicking of dUMP-containing DNA

Mahata

Molshanski-Mor

Goren

et al. 2021

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

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Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of ung and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.

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“…Deoxyuridine (dU) excision is an established strategy for generating 3' overhangs during molecular cloning and gene assembly procedures (4)(5)(6)(7)(8)(9). In this approach, dU nucleotides are excised from DNA through two enzymatic reactions: After the uracil base is excised by a uracil DNA glycosylase (10,11), the resulting abasic site is excised by an AP lyase such as endonuclease III (12,13) or endonuclease VIII (14,15). These reactions, which are readily performed together using a commercially available Uracil-Specific Excision Reagent (USER â ) enzyme mixture (New England Biolabs), generate a one nucleotide gap with a 5' phosphate that can be used to join DNA molecules.…”

Section: Introductionmentioning

confidence: 99%

Efficient linear dsDNA tagging using deoxyuridine excision

Strobel

2021

Preprint

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Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3′ ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT→dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3′ overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5′ overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.

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DNA N-glycosidases: properties of uracil-DNA glycosidase from Escherichia coli.

Cited by 504 publications

References 49 publications

Thermostable uracil-DNA glycosylase from Thermotoga maritima a member of a novel class of DNA repair enzymes

Thermostable uracil-DNA glycosylase from Thermotoga maritima a member of a novel class of DNA repair enzymes

A phage mechanism for selective nicking of dUMP-containing DNA

Efficient linear dsDNA tagging using deoxyuridine excision

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