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Mauro Magnani - One of the best experts on this subject based on the ideXlab platform.
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dexamethasone partially rescues ataxia telangiectasia mutated atm deficiency in ataxia telangiectasia by promoting a shortened protein variant retaining kinase activity
2012Co-Authors: Michele Menotta, Sara Biagiotti, Marzia Bianchi, Luciana Chessa, Mauro MagnaniAbstract:Abstract Ataxia Telangiectasia (AT) is a rare genetic disease still incurable, resulting from biallelic mutations in the Ataxia Telangiectasia-Mutated (ATM) gene. Recently, short-term treatment with glucocorticoid analogues improved neurological symptoms characteristic of this syndrome. Nevertheless, the molecular mechanism involved in glucocorticoid action in AT patients is not yet known. Here we describe, for the first time in mammalian cells, a short direct repeat-mediated non-canonical splicing event induced by dexamethasone, which leads to the skipping of mutations upstream of Nucleotide Residue 8450 of ATM coding sequence. The resulting transcript provides an alternative ORF translated in a new ATM variant with the complete kinase domain. This miniATM variant was also highlighted in lymphoblastoid cell lines from AT patients and resulted to be likely active. In conclusion, dexamethasone treatment may partly restore ATM activity in Ataxia Telangiectasia cells by a new molecular mechanism that overcomes most of the mutations so far described within this gene.
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dexamethasone partially rescues ataxia telangiectasia mutated atm deficiency in ataxia telangiectasia by promoting a shortened protein variant retaining kinase activity
2012Co-Authors: Michele Menotta, Sara Biagiotti, Marzia Bianchi, Luciana Chessa, Mauro MagnaniAbstract:Ataxia telangiectasia (AT) is a rare genetic disease, still incurable, resulting from biallelic mutations in the ataxia telangiectasia-mutated (ATM) gene. Recently, short term treatment with glucocorticoid analogues improved neurological symptoms characteristic of this syndrome. Nevertheless, the molecular mechanism involved in glucocorticoid action in AT patients is not yet known. Here we describe, for the first time in mammalian cells, a short direct repeat-mediated noncanonical splicing event induced by dexamethasone, which leads to the skipping of mutations upstream of Nucleotide Residue 8450 of ATM coding sequence. The resulting transcript provides an alternative ORF translated in a new ATM variant with the complete kinase domain. This miniATM variant was also highlighted in lymphoblastoid cell lines from AT patients and was shown to be likely active. In conclusion, dexamethasone treatment may partly restore ATM activity in ataxia telangiectasia cells by a new molecular mechanism that overcomes most of the mutations so far described within this gene.
Michele Menotta - One of the best experts on this subject based on the ideXlab platform.
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dexamethasone partially rescues ataxia telangiectasia mutated atm deficiency in ataxia telangiectasia by promoting a shortened protein variant retaining kinase activity
2012Co-Authors: Michele Menotta, Sara Biagiotti, Marzia Bianchi, Luciana Chessa, Mauro MagnaniAbstract:Abstract Ataxia Telangiectasia (AT) is a rare genetic disease still incurable, resulting from biallelic mutations in the Ataxia Telangiectasia-Mutated (ATM) gene. Recently, short-term treatment with glucocorticoid analogues improved neurological symptoms characteristic of this syndrome. Nevertheless, the molecular mechanism involved in glucocorticoid action in AT patients is not yet known. Here we describe, for the first time in mammalian cells, a short direct repeat-mediated non-canonical splicing event induced by dexamethasone, which leads to the skipping of mutations upstream of Nucleotide Residue 8450 of ATM coding sequence. The resulting transcript provides an alternative ORF translated in a new ATM variant with the complete kinase domain. This miniATM variant was also highlighted in lymphoblastoid cell lines from AT patients and resulted to be likely active. In conclusion, dexamethasone treatment may partly restore ATM activity in Ataxia Telangiectasia cells by a new molecular mechanism that overcomes most of the mutations so far described within this gene.
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dexamethasone partially rescues ataxia telangiectasia mutated atm deficiency in ataxia telangiectasia by promoting a shortened protein variant retaining kinase activity
2012Co-Authors: Michele Menotta, Sara Biagiotti, Marzia Bianchi, Luciana Chessa, Mauro MagnaniAbstract:Ataxia telangiectasia (AT) is a rare genetic disease, still incurable, resulting from biallelic mutations in the ataxia telangiectasia-mutated (ATM) gene. Recently, short term treatment with glucocorticoid analogues improved neurological symptoms characteristic of this syndrome. Nevertheless, the molecular mechanism involved in glucocorticoid action in AT patients is not yet known. Here we describe, for the first time in mammalian cells, a short direct repeat-mediated noncanonical splicing event induced by dexamethasone, which leads to the skipping of mutations upstream of Nucleotide Residue 8450 of ATM coding sequence. The resulting transcript provides an alternative ORF translated in a new ATM variant with the complete kinase domain. This miniATM variant was also highlighted in lymphoblastoid cell lines from AT patients and was shown to be likely active. In conclusion, dexamethasone treatment may partly restore ATM activity in ataxia telangiectasia cells by a new molecular mechanism that overcomes most of the mutations so far described within this gene.
Jimin Wang - One of the best experts on this subject based on the ideXlab platform.
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structural basis for differential insertion kinetics of dnmps opposite a difluorotoluene Nucleotide Residue
2012Co-Authors: Shuangluo Xia, William H Konigsberg, Soo Hyun Eom, Jimin WangAbstract:We have recently challenged the widely held view that 2,4-difluorotoluene (dF) is a nonpolar isosteric analogue of the Nucleotide dT, incapable of forming hydrogen bonds (HBs). To gain a further understanding for the kinetic preference that favors dAMP insertion opposite a templating dF, a result that mirrors the base selectivity that favors dAMP insertion opposite dT by RB69 DNA polymerase (RB69pol), we determined presteady-state kinetic parameters for incorporation of four dNMPs opposite dF by RB69pol and solved the structures of corresponding ternary complexes. We observed that both the F2 and F4 substituent of dF in these structures serve as HB acceptors forming HBs either directly with dTTP and dGTP or indirectly with dATP and dCTP via ordered water molecules. We have defined the shape and chemical features of each dF/dNTP pair in the RB69pol active site without the corresponding phosphodiester-linkage constraints of dF/dNs when they are embedded in isolated DNA duplexes. These features can explain t...
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hydrogen bonding capability of a templating difluorotoluene Nucleotide Residue in an rb69 dna polymerase ternary complex
2011Co-Authors: Shuangluo Xia, William H Konigsberg, Jimin WangAbstract:Results obtained using 2,4-difluorotoluene nucleobase (dF) as a nonpolar thymine isostere by Kool and colleagues challenged the Watson–Crick dogma that hydrogen bonds between complementary bases are an absolute requirement for accurate DNA replication. Here, we report crystal structure of an RB69 DNA polymerase L561A/S565G/Y567A triple mutant ternary complex with a templating dF opposite dTTP at 1.8 A-resolution. In this structure, direct hydrogen bonds were observed between: (i) dF and the incoming dTTP, (ii) dF and Residue G568 of the polymerase, and (iii) dF and ordered water molecules surrounding the nascent base pair. Therefore, this structure provides evidence that a templating dF can form novel hydrogen bonds with the incoming dTTP and with the enzyme that differ from those formed with a templating dT.
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insights into base selectivity from the 1 8 a resolution structure of an rb69 dna polymerase ternary complex
2011Co-Authors: Mina Wang, Shuangluo Xia, Gregor Blaha, Thomas A Steitz, William H Konigsberg, Jimin WangAbstract:DNA polymerases (pols)1 are essential components of replicases, which are multiprotein complexes responsible for copying genomes of all organisms with high fidelity (for reviews, see refs (1−3)). The mechanistic details of how these pols accomplish this task with such remarkable accuracy are not fully understood. RB69 DNA polymerase (RB69 pol) is the founding member of the B family of pols (4), as it was the first pol to have its structure determined in the apo form and then in a ternary complex (5,6). RB69 pol also shares sequence similarities with a number of eukaryotic pols, including human pols α, δ, and e (4). Structural studies of all these pols have provided tantalizing clues that continue to be explored as the resolution of ternary complex structures has improved. One of the hallmarks of DNA pol ternary complex structures is that Residues near the active site interact directly with the incoming dNTP as well as with the templating base in the minor groove of a DNA duplex, forming a nascent base pair-binding pocket (NBP) that is essential for recognition of Watson−Crick (W−C) base pairs. Occasionally, an incorrect incoming dNTP is mispaired with the templating base and incorporated onto the 3′ end of the growing primer strand. Even when this occurs, extension beyond this mismatched Nucleotide is seldom observed, and the mismatched Nucleotide Residue is nearly always removed by an exonuclease activity that may be present either in a separate domain of the pol or as a separate subunit of the replicase. However, most of the base discrimination occurs when an incoming dNTP enters the binary complex. The correct incoming dNTP triggers Fingers closing and stabilizes the closed ternary complex, allowing proper alignment of the substrates prior to nucleotidyl transfer. When an incorrect incoming dNTP triggers the open-to-closed transition, the resulting closed ternary complex is unstable. As a consequence, the rate of reopening is much faster than the rate of nucleotidyl transfer and the mismatched dNTP is released (3,7). Earlier studies have attempted to determine the differences between W−C and non-W−C base pairs in DNA duplexes (8,9). These studies have shown that mispairs distort the geometry of the duplex so that the cross-strand C1′−C1′ distances and the orientation of the glycosidic bonds as well as the positions of common N3 and O2 hydrogen bond acceptors in the minor groove of the duplex are altered relative to those of W−C base pairs. For example, the dT-dG mispair prefers a wobble geometry with the base of dG displaced toward the minor groove of the duplex. When DNA interacts with a pol, the minor groove of the bound DNA is widened near the primer/template (P/T) junction (10), which may also change preferences for mispaired geometries. Thus, depending on the specific interactions between the DNA and the pol at different locations in the P/T duplex, a dT-dG mispair can adopt either wobble or inverted wobble geometry with dG being displaced toward the minor or major groove (11). Because both wobble and inverted wobble forms of a dT-dG mispair have been observed, the energetic and steric differences for N3 and O2 hydrogen bond acceptors between a dT-dG mispair and a dC-dG pair are likely to be relatively small and unable to account for the large disparity in the kinetics exhibited by pols for insertion of dTMP versus dCMP opposite a templating dG. Thus, changes in the orientation of the glycosidic bonds as well as in the C1′−C1′ distances between mispaired and W−C base paired incoming dNTPs may play a greater role in base selectivity than the precise positioning of the N3 and O2 hydrogen bond acceptors. To identify the structural features of nascent base pairs that allow a pol to discriminate between correct and incorrect dNTPs, structures of ternary complexes are needed with both correct and incorrect nascent base pairs. Structures of a number of ternary complexes with correct dNTPs have been obtained (6,12−14). However, it has been a challenge to capture a pol ternary complex with an incorrect dNTP in the insertion site. With W−C base pairs, the structures of binary and ternary complexes have provided clues about the mechanism of base recognition. For example, the structure of a Taq pol binary complex shows that Residues in the NBP directly recognize N3 and O2 hydrogen acceptors in a widened minor groove of the DNA duplex (10). Minor groove widening has also been observed in complexes of other pols such as T7 pol and pol β (12,13,15), implying a common base recognition mechanism. The proper positioning of the N3 and O2 hydrogen bond acceptors in the minor groove of the DNA duplex is one of the features that distinguish W−C from non-W−C base pairs (8,9). In contrast, the same N3 and O2 recognition pattern of nascent base pairs is not observed in RB69 pol ternary complexes (6), suggesting that B family pols recognize the N3 and O2 hydrogen bond acceptors in a different manner. Nevertheless, a common feature among different families of pols is that the Fingers domain undergoes a conformational change after encountering correct dNTPs (6,13,14). This transition has been proposed as a checkpoint for discriminating against incorrect incoming dNTPs, although how this happens remains poorly understood. To address the base selectivity issue for RB69 pol, we have determined the crystal structure of its ternary complex with dCTP opposite a templating dG at 1.8 A resolution, which is significantly higher than our previous resolution of 2.6 A for a structure that had dTTP opposite dA (6). With the improved resolution, we have observed structural features that could not be seen previously and have confirmed many important inferences that were based on the 2.6 A structure, particularly the importance of three unusual nonpolar−polar interactions in the pol active site and the role of the newly observed hydration network in the incorporation of correct dNMPs. We have compared this 1.8 A resolution structure with the apo form of RB69 pol and provide additional insights into an open-to-closed conformational change of RB69 pol and its effects on the conformation of the incoming dCTP when it is part of the ternary complex. Finally, we have extended our comparison of the RB69 pol structure to the binary, ternary, and apo structures of ϕ29 DNA polymerase (ϕ29 pol) as well as to the ternary complex of yeast pol δ (16,17), in an attempt to identify the common features among the B family pols that are important for base selectivity.
Guang Hsiung Kou - One of the best experts on this subject based on the ideXlab platform.
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transcriptional analysis of the dna polymerase gene of shrimp white spot syndrome virus
2002Co-Authors: Lili Chen, Chunghsiung Wang, Han Ching Wang, Chiujung Huang, Shaoen Peng, Yengu Chen, Shinjen Lin, Wei Yu Chen, Changfeng Dai, Guang Hsiung KouAbstract:The white spot syndrome virus DNA polymerase (DNA pol) gene (WSSV dnapol) has already been tentatively identified based on the presence of highly conserved motifs, but it shows low overall homology with other DNA pols and is also much larger (2351 amino acid Residues vs 913-1244 aa). In the present study we perform a transcriptional analysis of the WSSV dnapol gene using the total RNA isolated from WSSV-infected shrimp at different times after infection. Northern blot analysis with a WSSV dnapol-specific riboprobe found a major transcript of 7.5 kb. 5-RACE revealed that the major transcription start point is located 27 Nucleotides downstream of the TATA box, at the Nucleotide Residue A within a CAGT motif, one of the initiator (Inr) motifs of arthropods. In a temporal expression analysis using differential RT-PCR, WSSV dnapol transcripts were detected at low levels at 2–4 h.p.i., increased at 6 h.p.i., and remained fairly constant thereafter. This is similar to the previously reported transcription patterns for genes encoding the key enzyme of Nucleotide metabolism, riboNucleotide reductase. Phylogenetic analysis showed that the DNA pols from three different WSSV isolates form an extremely tight cluster. In addition, similar to an earlier phylogenetic analysis of WSSV protein kinase, the phylogenetic tree of viral DNA pols further supports the suggestion that WSSV is a distinct virus (likely at the family level) that does not belong to any of the virus families that are currently recognized. © 2002 Elsevier Science (USA)
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transcriptional analysis of the riboNucleotide reductase genes of shrimp white spot syndrome virus
2000Co-Authors: Mengfeng Tsai, Marielle C W Van Hulten, Huey Fen Tzeng, Chih Ming Chou, Chang Jen Huang, Chunghsiung Wang, Jungyaw Lin, J M Vlak, Guang Hsiung KouAbstract:Abstract The causative agent of white spot syndrome (WSS) is a large double-stranded DNA virus, WSSV, which is probably a representative of a new genus, provisionally called Whispovirus. From previously constructed WSSV genomic libraries of a Taiwan WSSV isolate, clones with open reading frames (ORFs) that encode proteins with significant homology to the class I riboNucleotide reductase large (RR1) and small (RR2) subunits were identified. WSSV rr1 and rr2 potentially encode 848 and 413 amino acids, respectively. RNA was isolated from WSSV-infected shrimp at different times after infection and Northern blot analysis with rr1 - and rr2 -specific riboprobes found major transcripts of 2.8 and 1.4 kb, respectively. 5′ RACE showed that the major rr1 transcript started at a position of −84 (C) relative to the ATG translational start, while transcription of the rr2 gene started at Nucleotide Residue −68 (T). A consensus motif containing the transcriptional start sites for rr1 and rr2 was observed (TCAc/tTC). Northern blotting and RT-PCR showed that the transcription of rr1 and rr2 started 4–6 h after infection and continued for at least 60 h. The rr1 and rr2 genes thus appear to be WSSV “early genes.”
Sara Biagiotti - One of the best experts on this subject based on the ideXlab platform.
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dexamethasone partially rescues ataxia telangiectasia mutated atm deficiency in ataxia telangiectasia by promoting a shortened protein variant retaining kinase activity
2012Co-Authors: Michele Menotta, Sara Biagiotti, Marzia Bianchi, Luciana Chessa, Mauro MagnaniAbstract:Abstract Ataxia Telangiectasia (AT) is a rare genetic disease still incurable, resulting from biallelic mutations in the Ataxia Telangiectasia-Mutated (ATM) gene. Recently, short-term treatment with glucocorticoid analogues improved neurological symptoms characteristic of this syndrome. Nevertheless, the molecular mechanism involved in glucocorticoid action in AT patients is not yet known. Here we describe, for the first time in mammalian cells, a short direct repeat-mediated non-canonical splicing event induced by dexamethasone, which leads to the skipping of mutations upstream of Nucleotide Residue 8450 of ATM coding sequence. The resulting transcript provides an alternative ORF translated in a new ATM variant with the complete kinase domain. This miniATM variant was also highlighted in lymphoblastoid cell lines from AT patients and resulted to be likely active. In conclusion, dexamethasone treatment may partly restore ATM activity in Ataxia Telangiectasia cells by a new molecular mechanism that overcomes most of the mutations so far described within this gene.
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dexamethasone partially rescues ataxia telangiectasia mutated atm deficiency in ataxia telangiectasia by promoting a shortened protein variant retaining kinase activity
2012Co-Authors: Michele Menotta, Sara Biagiotti, Marzia Bianchi, Luciana Chessa, Mauro MagnaniAbstract:Ataxia telangiectasia (AT) is a rare genetic disease, still incurable, resulting from biallelic mutations in the ataxia telangiectasia-mutated (ATM) gene. Recently, short term treatment with glucocorticoid analogues improved neurological symptoms characteristic of this syndrome. Nevertheless, the molecular mechanism involved in glucocorticoid action in AT patients is not yet known. Here we describe, for the first time in mammalian cells, a short direct repeat-mediated noncanonical splicing event induced by dexamethasone, which leads to the skipping of mutations upstream of Nucleotide Residue 8450 of ATM coding sequence. The resulting transcript provides an alternative ORF translated in a new ATM variant with the complete kinase domain. This miniATM variant was also highlighted in lymphoblastoid cell lines from AT patients and was shown to be likely active. In conclusion, dexamethasone treatment may partly restore ATM activity in ataxia telangiectasia cells by a new molecular mechanism that overcomes most of the mutations so far described within this gene.
