The effect of sequence context on the activity of cytosine DNA glycosylases

2015 ◽  
Vol 11 (12) ◽  
pp. 3273-3278
Author(s):  
Scott T. Kimber ◽  
Tom Brown ◽  
Keith R. Fox
Keyword(s):  
Dna Glycosylase ◽  
Dna Glycosylases ◽  
Sequence Context ◽  
Uracil Dna Glycosylase

We have examined how sequence context affects the ability of (N204D:L272A) mutants of uracil DNA glycosylase to cleave CX mismatches.

Complexes of the uracil-DNA glycosylase inhibitor protein, Ugi, with Mycobacterium smegmatis and Mycobacterium tuberculosis uracil-DNA glycosylases

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1647-1658 ◽  
Author(s):  
Narottam Acharya ◽  
Pradeep Kumar ◽  
Umesh Varshney
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Three Dimensional ◽  
Model Organism ◽  
Dna Glycosylase ◽  
Structural Elements ◽  
Dna Glycosylases ◽  
Hydrophobic Pocket ◽  
Uracil Dna Glycosylase ◽  
Simplex Virus

Uracil, a promutagenic base, appears in DNA either by deamination of cytosine or by incorporation of dUMP by DNA polymerases. This unconventional base in DNA is removed by uracil-DNA glycosylase (UDG). Interestingly, a bacteriophage-encoded short polypeptide, UDG inhibitor (Ugi), specifically inhibits UDGs by forming a tight complex. Three-dimensional structures of the complexes of Ugi with UDGs from Escherichia coli, human and herpes simplex virus have shown that two of the structural elements in Ugi, the hydrophobic pocket and the β1-edge, establish key interactions with UDGs. In this report the characterization of complexes of Ugi with UDGs from Mycobacterium tuberculosis, a pathogenic bacterium, and Mycobacterium smegmatis, a widely used model organism for the former, is described. Unlike the E. coli (Eco) UDG-Ugi complex, which is stable to treatment with 8 M urea, the mycobacterial UDG-Ugi complexes dissociate in 5–6 M urea. Furthermore, the Ugi from the complexes of mycobacterial UDGs can be exchanged by the DNA substrate. Interestingly, while EcoUDG sequestered Ugi into the EcoUDG-Ugi complex when incubated with mycobacterial UDG-Ugi complexes, even a large excess of mycobacterial UDGs failed to sequester Ugi from the EcoUDG-Ugi complex. However, the M. tuberculosis (Mtu) UDG-Ugi complex was seen when MtuUDG was incubated with M. smegmatis (Msm) UDG-Ugi or EcoUDG(L191G)-Ugi complexes. The reversible nature of the complexes of Ugi with mycobacterial UDGs (which naturally lack some of the structural elements important for interaction with the β1-edge of Ugi) and with mutants of EcoUDG (which are deficient in interaction with the hydrophobic pocket of Ugi) highlights the significance of both classes of interaction in formation of UDG-Ugi complexes. Furthermore, it is shown that even though mycobacterial UDG-Ugi complexes dissociate in 5–6 M urea, Ugi is still a potent inhibitor of UDG activity in mycobacteria.

scholarly journals A New Class of Uracil–DNA Glycosylase Inhibitors Active against Human and Vaccinia Virus Enzyme

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6668
Author(s):  
Inga R. Grin ◽  
Grigory V. Mechetin ◽  
Rustem D. Kasymov ◽  
Evgeniia A. Diatlova ◽  
Anna V. Yudkina ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Viral Infections ◽  
Dna Glycosylase ◽  
Dna Glycosylases ◽  
Uracil Dna Glycosylase ◽  
Cytosine Deamination ◽  
Strand Breaks ◽  
Promising Target ◽  
Competitive Mechanism ◽  
Wide Range

Uracil–DNA glycosylases are enzymes that excise uracil bases appearing in DNA as a result of cytosine deamination or accidental dUMP incorporation from the dUTP pool. The activity of Family 1 uracil–DNA glycosylase (UNG) activity limits the efficiency of antimetabolite drugs and is essential for virulence in some bacterial and viral infections. Thus, UNG is regarded as a promising target for antitumor, antiviral, antibacterial, and antiprotozoal drugs. Most UNG inhibitors presently developed are based on the uracil base linked to various substituents, yet new pharmacophores are wanted to target a wide range of UNGs. We have conducted virtual screening of a 1,027,767-ligand library and biochemically screened the best hits for the inhibitory activity against human and vaccinia virus UNG enzymes. Although even the best inhibitors had IC50 ≥ 100 μM, they were highly enriched in a common fragment, tetrahydro-2,4,6-trioxopyrimidinylidene (PyO3). In silico, PyO3 preferably docked into the enzyme’s active site, and in kinetic experiments, the inhibition was better consistent with the competitive mechanism. The toxicity of two best inhibitors for human cells was independent of the presence of methotrexate, which is consistent with the hypothesis that dUMP in genomic DNA is less toxic for the cell than strand breaks arising from the massive removal of uracil. We conclude that PyO3 may be a novel pharmacophore with the potential for development into UNG-targeting agents.

scholarly journals Poxvirus uracil-DNA glycosylase-An unusual member of the family I uracil-DNA glycosylases

2016 ◽  
Vol 25 (12) ◽  
pp. 2113-2131 ◽  
Author(s):  
Norbert Schormann ◽  
Natalia Zhukovskaya ◽  
Gregory Bedwell ◽  
Manunya Nuth ◽  
Richard Gillilan ◽  
...  
Keyword(s):  
Dna Glycosylase ◽  
Dna Glycosylases ◽  
Uracil Dna Glycosylase ◽  
The Family

scholarly journals Repair of Hypoxanthine in DNA Revealed by DNA Glycosylases and Endonucleases From Hyperthermophilic Archaea

2021 ◽  
Vol 12 ◽  
Author(s):  
Tan Lin ◽  
Likui Zhang ◽  
Mai Wu ◽  
Donghao Jiang ◽  
Zheng Li ◽  
...  
Keyword(s):  
Excision Repair ◽  
Dna Glycosylase ◽  
Hyperthermophilic Archaea ◽  
Future Research ◽  
Dna Glycosylases ◽  
Uracil Dna Glycosylase ◽  
Alternative Pathways ◽  
Endonuclease V ◽  
Base Excision ◽  
Dna Strand

Since hyperthermophilic Archaea (HA) thrive in high-temperature environments, which accelerate the rates of deamination of base in DNA, their genomic stability is facing a severe challenge. Hypoxanthine (Hx) is one of the common deaminated bases in DNA. Generally, replication of Hx in DNA before repaired causes AT → GC mutation. Biochemical data have demonstrated that 3-methyladenine DNA glycosylase II (AlkA) and Family V uracil DNA glycosylase (UDG) from HA could excise Hx from DNA, thus triggering a base excision repair (BER) process for Hx repair. Besides, three endonucleases have been reported from HA: Endonuclease V (EndoV), Endonuclease Q (EndoQ), and Endonuclease NucS (EndoNucS), capable of cleaving Hx-containing DNA, thereby providing alternative pathways for Hx repair. Both EndoV and EndoQ could cleave one DNA strand with Hx, thus forming a nick and further initiating an alternative excision repair (AER) process for the follow-up repair. By comparison, EndoNucS cleaves both strands of Hx-containing DNA in a restriction endonuclease manner, thus producing a double-stranded break (DSB). This created DSB might be repaired by homologous recombination (HR) or by a combination activity of DNA polymerase (DNA pol), flap endonuclease 1 (FEN1), and DNA ligase (DNA lig). Herein, we reviewed the most recent advances in repair of Hx in DNA triggered by DNA glycosylases and endonucleases from HA, and proposed future research directions.

scholarly journals Uracil-DNA glycosylases preferentially excise mispaired uracil

1992 ◽  
Vol 287 (3) ◽  
pp. 1007-1010 ◽  
Author(s):  
A Verri ◽  
P Mazzarello ◽  
S Spadari ◽  
F Focher
Keyword(s):  
Substrate Specificity ◽  
Dna Glycosylase ◽  
Complementary Strand ◽  
Dna Glycosylases ◽  
Proliferating Cells ◽  
Uracil Dna Glycosylase ◽  
Cytosine Deamination ◽  
Efficient Recognition ◽  
Single Stranded Oligonucleotide

We have investigated the substrate specificity of human, viral and bacterial uracil-DNA glycosylases employing as substrate double-stranded oligonucleotides containing in the same position of the 5′-32P-labelled strand an uracil residue facing, on the complementary strand, guanine (mimicking cytosine deamination) or adenine (mimicking dUTP misincorporation). The enzyme removal of uracil was monitored and quantified by the generation of alkali-sensitive apyrimidinic sites. All three uracil-DNA glycosylases excise uracil from mispaired oligonucleotides (U/G) more efficiently than from paired oligonucleotides (U/A). The enzymes also remove uracil from single-stranded oligonucleotide with an efficiency similar to that observed with U/A paired oligonucleotide. The efficient recognition of U/G mispair by uracil-DNA glycosylase is important in minimizing miscoding transcripts and C/G-->T/A transitions in proliferating cells.

scholarly journals Functional Analysis on a Naturally Occurring Variant of the Staphylococcus Aureus Uracil DNA Glycosylase Inhibitor

2017 ◽  
Author(s):  
Veronika Papp-Kádár ◽  
Zoltán Balázs ◽  
Gergely N. Nagy ◽  
Tünde Juhász ◽  
Károly Liliom ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Complex Formation ◽  
Excision Repair ◽  
Dna Glycosylase ◽  
Dna Glycosylases ◽  
Uracil Dna Glycosylase ◽  
Naturally Occurring ◽  
Base Excision ◽  
Interaction Surface ◽  
Uracil Base

Repair of DNA damage relies on various pathways including the base excision repair (BER) which targets erroneous bases in the DNA. Here, Uracil-DNA glycosylases (UDGs) are responsible for recognition and removal of uracil base from the DNA. Here, we characterize the interaction of Staphylococcus aureus UDG (SAUDG) with a naturally occurring variant of S. aureus uracil-DNA glycosylase inhibitor (SAUGI). This variant contains a histidine instead of a glutamate at the 24th position which affects the SAUDG:SAUGI interaction surface. We assessed the complex formation of SAUDG with these two SAUGI variants by independent biophysical methods. Our data reveal that the residue difference at the 24th position does not have a marked effect on the binding affinity, yet it confers alteration of the thermodynamics of the interaction. We propose that the E24H variant of SAUGI allows efficient complex formation, and consequently, inhibition of SAUDG.

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