Expanding the DNA alphabet in the fruit fly

Horváth, András; Békési, Angéla; Muha, Villő; Erdélyi, Miklós; Vértessy, Beáta G.

doi:10.4161/fly.23192

Cited by 10 publications

(6 citation statements)

References 38 publications

(40 reference statements)

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“…Among these, dUTPase and uracil DNA glycosylase enzymes are responsible for keeping DNA uracil-free [1][2][3]. Absence of dUTPase (in differentiated tissues, knock-out models or several microorganisms) [4][5][6][7][8][9] or its inhibition combined with thymidylate synthase-targeted chemotherapies [10,11] is expected to lead to an increased level of uracil in DNA. Under such circumstances, DNA polymerases may readily incorporate uracil into DNA and uracil excision repair is activated.…”

Section: Introductionmentioning

confidence: 99%

The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition

et al. 2019

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Protein inhibitors of key DNA repair enzymes play an important role in deciphering physiological pathways responsible for genome integrity, and may also be exploited in biomedical research. The staphylococcal repressor StlSaPIbov1 protein was described to be an efficient inhibitor of dUTPase homologues showing a certain degree of species-specificity. In order to provide insight into the inhibition mechanism, in the present study we investigated the interaction of StlSaPIbov1 and Escherichia coli dUTPase. Although we observed a strong interaction of these proteins, unexpectedly the E. coli dUTPase was not inhibited. Seeking a structural explanation for this phenomenon, we identified a key amino acid position where specific mutations sensitized E. coli dUTPase to StlSaPIbov1 inhibition. We solved the three-dimensional (3D) crystal structure of such a mutant in complex with the substrate analogue dUPNPP and surprisingly found that the C-terminal arm of the enzyme, containing the P-loop-like motif was ordered in the structure. This segment was never localized before in any other E. coli dUTPase crystal structures. The 3D structure in agreement with solution phase experiments suggested that ordering of the flexible C-terminal segment upon substrate binding is a major factor in defining the sensitivity of E. coli dUTPase for StlSaPIbov1 inhibition.

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Section: Introductionmentioning

confidence: 99%

The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition

et al. 2019

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“…The surprisingly high uracil content (estimated as >25 000 uracil/million bases) of reverse-transcribed HIV genomic DNA has been suggested to play an important role in the viral life cycle ( 34 ). DNA from fruit fly larvae and pupae also contains highly elevated levels of uracil (200–2000 uracil/million bases) ( 35 , 36 ).…”

Section: Introductionmentioning

confidence: 99%

Detection of uracil within DNA using a sensitive labeling method forin vitroand cellular applications

Róna¹,

Scheer²,

Nagy³

et al. 2015

Nucleic Acids Res

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The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell.

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“…Developmental patterns in these organisms show strong correlation between the presence of uracil in their genomes and the lowered level of expression of dUTPase, an enzyme that hydrolyses dUTP to dUMP to avoid its incorporation in DNA. Thus, the lack of family 1 UDG (UNG) activity in these organisms is physiologically relevant in allowing occurrence of uracil in DNA ( 66 ). Whether the lack of highly efficient family 1 UDG in B. diazoefficiens contributes to its specialized physiology during nodulation or otherwise is not known.…”

Section: Discussionmentioning

confidence: 99%

Uracil DNA glycosylase (UDG) activities in Bradyrhizobium diazoefficiens: characterization of a new class of UDG with broad substrate specificity

Chembazhi

Patil

Sah

et al. 2017

Nucleic Acids Research

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Repair of uracils in DNA is initiated by uracil DNA glycosylases (UDGs). Family 1 UDGs (Ung) are the most efficient and ubiquitous proteins having an exquisite specificity for uracils in DNA. Ung are characterized by motifs A (GQDPY) and B (HPSPLS) sequences. We report a novel dimeric UDG, Blr0248 (BdiUng) from Bradyrhizobium diazoefficiens. Although BdiUng contains the motif A (GQDPA), it has low sequence identity to known UDGs. BdiUng prefers single stranded DNA and excises uracil, 5-hydroxymethyl-uracil or xanthine from it. BdiUng is impervious to inhibition by AP DNA, and Ugi protein that specifically inhibits family 1 UDGs. Crystal structure of BdiUng shows similarity with the family 4 UDGs in its overall fold but with family 1 UDGs in key active site residues. However, instead of a classical motif B, BdiUng has a uniquely extended protrusion explaining the lack of Ugi inhibition. Structural and mutational analyses of BdiUng have revealed the basis for the accommodation of diverse substrates into its substrate binding pocket. Phylogenetically, BdiUng belongs to a new UDG family. Bradyrhizobium diazoefficiens presents a unique scenario where the presence of at least four families of UDGs may compensate for the absence of an efficient family 1 homologue.

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Expanding the DNA alphabet in the fruit fly

Cited by 10 publications

References 38 publications

The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition

The Role of a Key Amino Acid Position in Species-Specific Proteinaceous dUTPase Inhibition

Detection of uracil within DNA using a sensitive labeling method forin vitroand cellular applications

Uracil DNA glycosylase (UDG) activities in Bradyrhizobium diazoefficiens: characterization of a new class of UDG with broad substrate specificity

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