Uracil

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Laurence H. Pearl - One of the best experts on this subject based on the ideXlab platform.

  • structural basis for Uracil recognition by archaeal family b dna polymerases
    Nature Structural & Molecular Biology, 2002
    Co-Authors: Mark J Fogg, Laurence H. Pearl, Bernard A Connolly
    Abstract:

    Deamination of cytosine to Uracil in a G-C base pair is a major promutagenic event, generating G-C→A-T mutations if not repaired before DNA replication. Archaeal family B DNA polymerases are uniquely able to recognize unrepaired Uracil in a template strand and stall polymerization upstream of the lesion, thereby preventing the irreversible fixation of an A-T mutation. We have now identified a 'pocket' in the N-terminal domains of archaeal DNA polymerases that is positioned to interact with the template strand and provide this ability. The structure of this pocket provides interacting groups that discriminate Uracil from the four normal DNA bases (including thymine). These groups are conserved in archaeal polymerases but absent from homologous viral polymerases that are unable to recognize Uracil. Using site-directed mutagenesis, we have confirmed the biological role of this pocket and have engineered specific mutations in the Pfu polymerase that confer the ability to read through template-strand Uracils and carry out PCR with dUTP in place of dTTP.

  • structure and function in the Uracil dna glycosylase superfamily
    Mutation Research-dna Repair, 2000
    Co-Authors: Laurence H. Pearl
    Abstract:

    Abstract Deamination of cytosine to Uracil is one of the major pro-mutagenic events in DNA, causing G:C→A:T transition mutations if not repaired before replication. Repair of Uracil-DNA is achieved in a base-excision pathway initiated by a Uracil-DNA glycosylase (UDG) enzyme of which four families have so far been identified. Family-1 enzymes are active against Uracil in ssDNA and dsDNA, and recognise Uracil explicitly in an extrahelical conformation via a combination of protein and bound-water interactions. Extrahelical recognition requires an efficient process of substrate location by ‘base-sampling’ probably by hopping or gliding along the DNA. Family-2 enzymes are mismatch specific and explicitly recognise the widowed guanine on the complementary strand rather than the extrahelical scissile pyrimidine. This allows a broader specificity so that some Family-2 enzymes can excise Uracil and 3,N4-ethenocytosine from mismatches with guanine. Although structures are not yet available for Family-3 (SMUG) and Family-4 enzymes, sequence analysis suggests similar overall folds, and identifies common active site motifs but with a surprising lack of conservation of catalytic residues between members of the super-family.

  • crystal structure of a g t u mismatch specific dna glycosylase mismatch recognition by complementary strand interactions
    Cell, 1998
    Co-Authors: T E Barrett, Laurence H. Pearl, Renos Savva, Tom Brown, George Panayotou, T Barlow, Josef Jiricny
    Abstract:

    G:U mismatches resulting from deamination of cytosine are the most common promutagenic lesions occurring in DNA. Uracil is removed in a base-excision repair pathway by Uracil DNA-glycosylase (UDG), which excises Uracil from both single- and double-stranded DNA. Recently, a biochemically distinct family of DNA repair enzymes has been identified, which excises both Uracil and thymine, but only from mispairs with guanine. Crystal structures of the mismatch-specific Uracil DNA-glycosylase (MUG) from E. coli, and of a DNA complex, reveal a remarkable structural and functional homology to UDGs despite low sequence identity. Details of the MUG structure explain its thymine DNA-glycosylase activity and the specificity for G:U/T mispairs, which derives from direct recognition of guanine on the complementary strand.

  • the structural basis of specific base excision repair by Uracil dna glycosylase
    Nature, 1996
    Co-Authors: Renos Savva, Tom Brown, Katherine E Mcauleyhecht, Laurence H. Pearl
    Abstract:

    The 1.75-A crystal structure of the Uracil-DNA glycosylase from herpes simplex virus type-1 reveals a new fold, distantly related to dinucleotide-binding proteins. Complexes with a trideoxynucleotide, and with Uracil, define the DNA-binding site and allow a detailed understanding of the exquisitely specific recognition of Uracil in DNA. The overall structure suggests binding models for elongated single- and double-stranded DNA substrates. Conserved residues close to the Uracil-binding site suggest a catalytic mechanism for hydrolytic base excision.

James T. Stivers - One of the best experts on this subject based on the ideXlab platform.

  • n terminal domain of human Uracil dna glycosylase hung2 promotes targeting to Uracil sites adjacent to ssdna dsdna junctions
    Nucleic Acids Research, 2018
    Co-Authors: Brian P Weiser, Gaddiel Rodriguez, Philip A Cole, James T. Stivers
    Abstract:

    The N-terminal domain (NTD) of nuclear human Uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward Uracils located 21 bp or less from the ssDNA-dsDNA junction. In the absence of RPA, hUNG2 still showed an 8-fold excision bias for Uracil located <10 bp from the junction, but only when the overhang had a 5' end. Biased targeting required the NTD and was not observed with the hUNG2 catalytic domain alone. Consistent with this requirement, the isolated NTD was found to bind weakly to ssDNA. These findings indicate that the NTD of hUNG2 targets the enzyme to ssDNA-dsDNA junctions using RPA-dependent and RPA-independent mechanisms. This structure-based specificity may promote efficient removal of Uracils that arise from dUTP incorporation during DNA replication, or additionally, Uracils that arise from DNA cytidine deamination at transcriptional R-loops during immunoglobulin class-switch recombination.

  • DNA translocation by human Uracil DNA glycosylase: the case of single-stranded DNA and clustered Uracils.
    Biochemistry, 2013
    Co-Authors: Joseph D. Schonhoft, James T. Stivers
    Abstract:

    Human Uracil DNA glycosylase (hUNG) plays a central role in DNA repair and programmed mutagenesis of Ig genes, requiring it to act on sparsely or densely spaced Uracil bases located in a variety of contexts, including U/A and U/G base pairs, and potentially Uracils within single-stranded DNA (ssDNA). An interesting question is whether the facilitated search mode of hUNG, which includes both DNA sliding and hopping, changes in these different contexts. Here we find that hUNG uses an enhanced local search mode when it acts on Uracils in ssDNA, and also, in a context where Uracils are densely clustered in duplex DNA. In the context of ssDNA, hUNG performs an enhanced local search by sliding with a mean sliding length larger than that of double-stranded DNA (dsDNA). In the context of duplex DNA, insertion of high-affinity abasic product sites between two Uracil lesions serves to significantly extend the apparent sliding length on dsDNA from 4 to 20 bp and, in some cases, leads to directionally biased 3′ → 5′ ...

  • Uracil dna glycosylase uses dna hopping and short range sliding to trap extrahelical Uracils
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Rishi H Porecha, James T. Stivers
    Abstract:

    The astonishingly efficient location and excision of damaged DNA bases by DNA repair glycosylases is an especially intriguing problem in biology. One example is the enzyme Uracil DNA glycosylase (UNG), which captures and excises rare extrahelical Uracil bases that have emerged from the DNA base stack by spontaneous base pair breathing motions. Here, we explore the efficiency and mechanism by which UNG executes intramolecular transfer and excision of two Uracil sites embedded on the same or opposite DNA strands at increasing site spacings. The efficiency of intramolecular site transfer decreased from 41 to 0% as the base pair spacing between Uracil sites on the same DNA strand increased from 20 to 800 bp. The mechanism of transfer is dominated by DNA hopping between landing sites of ≈10 bp size, over which rapid 1D scanning likely occurs. Consistent with DNA hopping, site transfer at 20- and 56-bp spacings was unaffected by whether the Uracils were placed on the same or opposite strands. Thus, UNG uses hopping and 3D diffusion through bulk solution as the principal pathways for efficient patrolling of long genomic DNA sequences for damage. Short-range sliding over the range of a helical turn allows for redundant inspection of very local DNA sequences and trapping of spontaneously emerging extrahelical Uracils.

  • enzymatic capture of an extrahelical thymine in the search for Uracil in dna
    Nature, 2007
    Co-Authors: Jared B Parker, M A Bianchet, Daniel J Krosky, Joshua I Friedman, Mario L Amzel, James T. Stivers
    Abstract:

    The enzyme Uracil DNA glycosylase (UNG) excises unwanted Uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of Uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U•A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U•A or U•G base pairs in a background of approximately 109 T•A or C•G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and Uracil is initiated by thermally induced opening of T•A and U•A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for Uracil, and may be involved in initial damage recognition by other DNA repair glycosylases. Uracil (U) belongs in RNA, where it takes the place filled by thymine in DNA. But if Uracil appears in DNA in error, it can lead to potentially life-threatening mutations. This can typically occur by chemical modification of cytosine. To counter this threat, cells use the enzyme Uracil DNA glycosylase to remove Uracil from DNA. The detailed mechanism by which this enzyme polices DNA for stray Uracils is now revealed. The DNA helix is not static but in a process rather like molecular 'breathing'; base pairs separate briefly then reform. When a Uracil base pops out of the helix it is grabbed by Uracil DNA glycosylase and removed. Thymine, differing only in one methyl group from Uracil, is similarly grabbed but as it does not quite fit the policing enzyme's active site, it is released to go about its business in the DNA molecule. The enzyme Uracil DNA glycosylase does not actively extrude just the Uracil base from the DNA helix to facilitate its removal; instead, transient, passive opening of thymine: adenine and Uracil: adenine base pairs allows both thymine and Uracil to become extrahelical, but only Uracil can subsequently fit in the active site.

  • Uracil-Directed Ligand Tethering: An Efficient Strategy for Uracil DNA Glycosylase (UNG) Inhibitor Development
    Journal of the American Chemical Society, 2005
    Co-Authors: Yu Lin Jiang, Daniel J Krosky, Lauren Seiple, James T. Stivers
    Abstract:

    Uracil DNA glycosylase (UNG) is an important DNA repair enzyme that recognizes and excises Uracil bases in DNA using an extrahelical recognition mechanism. It is emerging as a desirable target for small-molecule inhibitors given its key role in a wide range of biological processes including the generation of antibody diversity, DNA replication in a number of viruses, and the formation of DNA strand breaks during anticancer drug therapy. To accelerate the discovery of inhibitors of UNG we have developed a Uracil-directed ligand tethering strategy. In this efficient approach, a Uracil aldehyde ligand is tethered via alkyloxyamine linker chemistry to a diverse array of aldehyde binding elements. Thus, the mechanism of extrahelical recognition of the Uracil ligand is exploited to target the UNG active site, and alkyloxyamine linker tethering is used to randomly explore peripheral binding pockets. Since no compound purification is required, this approach rapidly identified the first small-molecule inhibitors o...

Renos Savva - One of the best experts on this subject based on the ideXlab platform.

  • crystal structure of a g t u mismatch specific dna glycosylase mismatch recognition by complementary strand interactions
    Cell, 1998
    Co-Authors: T E Barrett, Laurence H. Pearl, Renos Savva, Tom Brown, George Panayotou, T Barlow, Josef Jiricny
    Abstract:

    G:U mismatches resulting from deamination of cytosine are the most common promutagenic lesions occurring in DNA. Uracil is removed in a base-excision repair pathway by Uracil DNA-glycosylase (UDG), which excises Uracil from both single- and double-stranded DNA. Recently, a biochemically distinct family of DNA repair enzymes has been identified, which excises both Uracil and thymine, but only from mispairs with guanine. Crystal structures of the mismatch-specific Uracil DNA-glycosylase (MUG) from E. coli, and of a DNA complex, reveal a remarkable structural and functional homology to UDGs despite low sequence identity. Details of the MUG structure explain its thymine DNA-glycosylase activity and the specificity for G:U/T mispairs, which derives from direct recognition of guanine on the complementary strand.

  • the structural basis of specific base excision repair by Uracil dna glycosylase
    Nature, 1996
    Co-Authors: Renos Savva, Tom Brown, Katherine E Mcauleyhecht, Laurence H. Pearl
    Abstract:

    The 1.75-A crystal structure of the Uracil-DNA glycosylase from herpes simplex virus type-1 reveals a new fold, distantly related to dinucleotide-binding proteins. Complexes with a trideoxynucleotide, and with Uracil, define the DNA-binding site and allow a detailed understanding of the exquisitely specific recognition of Uracil in DNA. The overall structure suggests binding models for elongated single- and double-stranded DNA substrates. Conserved residues close to the Uracil-binding site suggest a catalytic mechanism for hydrolytic base excision.

Michael Emerman - One of the best experts on this subject based on the ideXlab platform.

  • Uracil dna glycosylase is dispensable for human immunodeficiency virus type 1 replication and does not contribute to the antiviral effects of the cytidine deaminase apobec3g
    Journal of Virology, 2006
    Co-Authors: Shari M Kaiser, Michael Emerman
    Abstract:

    It is well established that many host factors are involved in the replication of human immunodeficiency virus (HIV) type 1. One host protein, Uracil DNA glycosylase 2 (UNG2), binds to multiple viral proteins and is packaged into HIV type 1 virions. UNG initiates the removal of Uracils from DNA, and this has been proposed to be important both for reverse transcription and as a mediator to the antiviral effect of virion-incorporated Apobec3G, a cytidine deaminase that generates numerous Uracils in the viral DNA during virus replication. We used a natural human UNG−/− cell line as well as cells that express a potent catalytic active-site inhibitor of UNG to assess the effects of removing UNG activity on HIV infectivity. In both cases, we find UNG2 activity and protein to be completely dispensable for virus replication. Moreover, we find that virion-associated UNG2 does not affect the loss of infectivity caused by Apobec3G.

Federico Focher - One of the best experts on this subject based on the ideXlab platform.

  • Herpes simplex virus type 1 Uracil-DNA glycosylase: isolation and selective inhibition by novel Uracil derivatives.
    The Biochemical journal, 1993
    Co-Authors: Federico Focher, Silvio Spadari, Alessandro Verri, R. Manservigi, Joseph Gambino, George E. Wright
    Abstract:

    We have purified Herpes simplex type 1 (HSV1) Uracil-DNA glycosylase from the nuclei of HSV1-infected HeLa cells harvested 8 h post-infection, at which time the induction of the enzyme is a maximum. The enzyme has been shown to be distinct from the host enzyme, isolated from HeLa cells, by its lack of sensitivity to a monoclonal antibody to human Uracil-DNA glycosylase. Furthermore, several Uracil analogues were synthesized and screened for their capacity to discriminate between the viral and human Uracil-DNA glycosylases. Both enzymes were inhibited by 6-(p-alkylanilino)Uracils, but the viral enzyme was significantly more sensitive than the HeLa enzyme to most analogues. Substituents providing the best inhibitors of HSV1 Uracil-DNA glycosylase were found to be in the order: p-n-butyl < p-n-pentl = p-n-hexyl < p-n-heptyl < p-n-octyl. The most potent HSV1 enzyme inhibitor, 6-(p-n-octylanilino)Uracil (OctAU), with an IC50 of 8 microM, was highly selective for the viral enzyme. Short-term [3H]thymidine incorporation into the DNA of HeLa cells in culture was partially inhibited by OctAU, whereas it was unchanged when 6-(p-n-hexylanilino)Uracil was present at concentrations that completely inhibited HSV1 Uracil-DNA glycosylase activity. These compounds represent the first class of inhibitors that inhibit HSV1 Uracil-DNA glycosylase at concentrations in the micromolar range. The results suggest their possible use to evaluate the functional role of HSV1 Uracil-DNA glycosylase in viral infections and re-activation in nerve cells.

  • Herpes simplex virus type 1 Uracil-DNA glycosylase: isolation and selective inhibition by novel Uracil derivatives.
    Biochemical Journal, 1993
    Co-Authors: Federico Focher, Silvio Spadari, Alessandro Verri, R. Manservigi, Joseph Gambino, George E. Wright
    Abstract:

    We have purified Herpes simplex type 1 (HSV1) Uracil-DNA glycosylase from the nuclei of HSV1-infected HeLa cells harvested 8 h post-infection, at which time the induction of the enzyme is a maximum. The enzyme has been shown to be distinct from the host enzyme, isolated from HeLa cells, by its lack of sensitivity to a monoclonal antibody to human Uracil-DNA glycosylase. Furthermore, several Uracil analogues were synthesized and screened for their capacity to discriminate between the viral and human Uracil-DNA glycosylases. Both enzymes were inhibited by 6-(p-alkylanilino)Uracils, but the viral enzyme was significantly more sensitive than the HeLa enzyme to most analogues. Substituents providing the best inhibitors of HSV1 Uracil-DNA glycosylase were found to be in the order: p-n-butyl

  • Uracil-DNA glycosylases preferentially excise mispaired Uracil.
    Biochemical Journal, 1992
    Co-Authors: Alessandro Verri, Paolo Mazzarello, Silvio Spadari, Federico Focher
    Abstract:

    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.