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The Application of Fragment-based Approaches to the Discovery of Drugs for Neglected Tropical Diseases
Published in Venkatesan Jayaprakash, Daniele Castagnolo, Yusuf Özkay, Medicinal Chemistry of Neglected and Tropical Diseases, 2019
Christina Spry, Anthony G. Coyne
Uracil-DNA glycosylase (UDG) catalyses excision of misincorporated uracil from DNA and initiates “base-excision repair” (Pena-Diaz et al. 2004). Having solved the structure of a putative uracil-DNA glycosylase from Leishmania naiffi, crystals of the enzyme were soaked with 68 cocktails each containing ten fragments. Four fragments were observed to bind to the enzyme (Table 2), as was DMSO. A derivative of one fragment hit (5-chloro-2-methoxybenzoic acid) was designed and found to inhibit the activity of the enzyme 1000-fold more potently than the parent fragment (IC50 = 15 μM (LE = 0.44) vs 15 mM (LE = 0.21); Table 2). Recently, and since this fragment screen, Mishra et al. (2018) have shown that expression of the gene coding for UDG is upregulated in Leishmania donovani in response to treatment with commonly used antileishmanial drugs, and that drug-resistant clinical isolates of L. donovani show higher levels of the transcript encoding UDG. For these reasons, the enzyme has been proposed as a potential target for new combination therapies. The hits identified here may therefore serve as good starting points for development of tool compounds/new drugs targeting this enzyme.
Target Amplification-Based Techniques
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
Antoinette A.T.P. Brink, Peter J.F. Snijders, Chris J.L.M. Meijer
Uracil DNA glycosylase (UDG) cleaves the uracil from the phosphodiester backbone of uracil-containing DNA. The resulting apyrimidinic sites block replication by DNA polymerases and are very labile to acid–base hydrolysis. UDG does not react with free deoxyuridine 5-triphosphate (dUTP) and is inactivated by heat denaturation. These properties can be utilized to prevent reaction product carry-over by incorporating dUTP in all PCR products (by substituting dUTP for dTTP or by incorporating uracil during primer synthesis) and treating all subsequent preassembled reactions with UDG, followed by heat-denaturation of UDG.39 UDG is commercially available (e.g., AmpErase from Applied Biosystems) and is used in many diagnostic laboratories.
Unfolding the Role of Splicing Factors and RNA Debranching in AID Mediated Antibody Diversification
Published in International Reviews of Immunology, 2021
Ankit Jaiswal, Amit Kumar Singh, Anubhav Tamrakar, Prashant Kodgire
SHM takes place in the variable regions of Ig light and heavy chain in B-cell, upon antigenic stimulation [6]. SHM introduces point mutation in the variable region of Ig genes which produces a high as well as a low-affinity antibody. Further, B-cells expressing high-affinity antibody expands into plasma B-cell and memory B-cell, whereas apoptosis of B-cells expressing low affinity takes place. SHM together with clonal selection is known as affinity maturation [7]. SHM takes place in the dark zone (DZ) of the germinal center. Subsequently, SHM generated antibody moves to the light zone (LZ) of the germinal center where the clonal selection of high-affinity antibody and CSR takes place. SHM is mediated by genome mutator enzyme AID, that induced a point mutation via deamination of cytosine (C) into uracil (U), C to U conversion leads to the creation of a mismatch that can have various fates inside the B-cell [8]. If a U:G mismatch is unrepaired, and replication occurs then it leads to the creation of a transition mutation from C to T [9]. Moreover, if Uracil DNA glycosylase (UNG) recognize U:G mismatch, it removes uracil base, leads to the creation of abasic site (AP) [10]. In the context of Ig genes, AP site is repaired by the recruitment of error-prone DNA polymerase results in a mutation. Additionally, if U:G mismatch is processed by base excision repair (BER) or mismatch repair system (MER) results in insertion, deletion as well as substitution mutation [11] (Figure 1A). Thus, AID initiated mutations in Ig genes are unfaithfully repaired that result in SHM.
More data on ancient human mitogenome variability in Italy: new mitochondrial genome sequences from three Upper Palaeolithic burials
Published in Annals of Human Biology, 2021
Alessandra Modi, Stefania Vai, Cosimo Posth, Chiara Vergata, Valentina Zaro, Maria Angela Diroma, Francesco Boschin, Giulia Capecchi, Stefano Ricci, Annamaria Ronchitelli, Giulio Catalano, Gabriele Lauria, Giuseppe D'Amore, Luca Sineo, David Caramelli, Martina Lari
NGS libraries were prepared starting from 20 µl of DNA extract for each specimen following a double-stranded DNA protocol optimised for ancient samples, in order to make the DNA immortalised, barcoded and available for the Next Generation Sequencing (NGS) on Illumina platforms. A partial uracil-DNA-glycosylase treatment (Rohland et al. 2015) was performed for ST2 sample. This treatment removes uracile residues and abasic sites in the internal portion of the molecules, but partially preserves the characteristic deamination pattern associated with aDNA damage at the ends of the fragments, which can be used to discriminate endogenous DNA from possible modern contaminants (Rohland et al. 2015). No uracil-DNA glycosylase treatment was performed for AC16 and PA12 samples (Meyer and Kircher 2010). A unique combination of two indexes per specimen was used for barcoding. Libraries were enriched for mtDNA following a multiplexed capture protocol (Maricic et al. 2010) and sequenced on an Illumina MiSeq v3 2 × 75 + 8 + 8 bp chemistry. For ST2, the enriched library was sequenced on Illumina NovaSeq 6000, setting 1 × 100 + 8 + 8 run parameters.
AID Biology: A pathological and clinical perspective
Published in International Reviews of Immunology, 2018
Meenal Choudhary, Anubhav Tamrakar, Amit Kumar Singh, Monika Jain, Ankit Jaiswal, Prashant Kodgire
Different enzymes including activation-induced cytidine deaminase (AID) and Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, Rad51D) are involved in the process of gene conversion. AID-initiated uracils are excised by uracil DNA glycosylase (UNG) creating an abasic site resulting in DNA lesions. Expression of an inhibitor for UNG in DT40 cells diminished the rate of gene conversion [52]. The ensuing study revealed that gene conversion proceeds through single or double-stranded DNA breaks at the AID-dependent uracils [53]. Further, the UNG-deficient mutants show a complete block in gene conversion along with the accumulation of transition mutations within the rearranged IgL chain gene [54]. In addition, ablation of the RAD51 paralogues (XRCC2, XRCC3 or RAD51B) in the chicken DT40 cells resulted in a shift from gene conversion to somatic hypermutation [55]. Moreover, a stepwise deletion of the ψV genes at the rearranged light chain locus abrogates gene conversion, thereby triggering AID-induced hypermutation in DT40 [56].