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Myron F Goodman - One of the best experts on this subject based on the ideXlab platform.
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AID and Apobec3G haphazard deamination and mutational diversity
Cellular and Molecular Life Sciences, 2013Co-Authors: Malgorzata Jaszczur, Phuong Pham, Jeffrey G. Bertram, Matthew D. Scharff, Myron F GoodmanAbstract:Activation-induced deoxycytidine Deaminase (AID) and Apobec 3G (Apo3G) cause mutational diversity by initiating mutations on regions of single-stranded (ss) DNA. Expressed in B cells, AID deaminates C → U in actively transcribed immunoglobulin (Ig) variable and switch regions to initiate the somatic hypermutation (SHM) and class switch recombination (CSR) that are essential for antibody diversity. Apo3G expressed in T cells catalyzes C deaminations on reverse transcribed cDNA causing HIV-1 retroviral inactivation. When operating properly, AID- and Apo3G-initiated mutations boost human fitness. Yet, both enzymes are potentially powerful somatic cell “mutators”. Loss of regulated expression and proper genome targeting can cause human cancer. Here, we review well-established biological roles of AID and Apo3G. We provide a synopsis of AID partnering proteins during SHM and CSR, and describe how an Apo2 crystal structure provides “surrogate” insight for AID and Apo3G biochemical behavior. However, large gaps remain in our understanding of how dC Deaminases search ssDNA to identify trinucleotide motifs to deaminate. We discuss two recent methods to analyze ssDNA scanning and deamination. Apo3G scanning and deamination is visualized in real-time using single-molecule FRET, and AID deamination efficiencies are determined with a random walk analysis. AID and Apo3G encounter many candidate deamination sites while scanning ssDNA. Generating mutational diversity is a principal aim of AID and an important ancillary property of Apo3G. Success seems likely to involve hit and miss deamination motif targeting, biased strongly toward miss.
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apobec3g dna Deaminase acts processively 3 5 on single stranded dna
Nature Structural & Molecular Biology, 2006Co-Authors: Linda Chelico, Phuong Pham, Peter Calabrese, Myron F GoodmanAbstract:Akin to a 'Trojan horse,' APOBEC3G DNA Deaminase is encapsulated by the HIV virion. APOBEC3G facilitates restriction of HIV-1 infection in T cells by deaminating cytosines in nascent minus-strand complementary DNA. Here, we investigate the biochemical basis for C → U targeting. We observe that APOBEC3G binds randomly to single-stranded DNA, then jumps and slides processively to deaminate target motifs. When confronting partially double-stranded DNA, to which APOBEC3G cannot bind, sliding is lost but jumping is retained. APOBEC3G shows catalytic orientational specificity such that deamination occurs predominantly 3′ → 5′ without requiring hydrolysis of a nucleotide cofactor. Our data suggest that the G → A mutational gradient generated in viral genomic DNA in vivo could result from an intrinsic processive directional attack by APOBEC3G on single-stranded cDNA.
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methylation protects cytidines from aid mediated deamination
Molecular Immunology, 2005Co-Authors: Mani Larijani, Myron F Goodman, Matthew D. Scharff, Darina Frieder, Timothy Michael Sonbuchner, Ronda Bransteitter, Eric E Bouhassira, Alberto MartinAbstract:Somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion of immunoglobulin genes require activation-induced cytidine Deaminase (AID). AID initiates these events by deaminating cytidines within antibody variable and switch regions. The mechanism that restricts mutation to antibody genes is not known. Although genes other than antibody genes have been found to mutate, not all highly transcribed genes mutate. Thus, somatic hypermutation does not target all genes and suggests a mechanism that either recruits AID to genes for mutation, and/or one that protects genes from promiscuous AID activity. Recent evidence suggests that AID deaminates methyl cytidines inefficiently. Methylation of cytidines could thus represent a means to protect the genome from potentially harmful AID activity that occurs outside of the immunoglobulin loci. To test this premise, we examined whether AID could deaminate methylated-CpG motifs in different sequence contexts. In agreement with a report that suggests that AID has processive-like properties in vitro, we found that AID could completely deaminate single-stranded DNA tracks in plasmid substrates that were greater than 300 nucleotides in length. In addition, methylated-CpG motifs, but not their unmethylated counterparts, were protected from AID-mediated deamination. However, methylation did not protect cytidines that neighbored CpG motifs indicating that methylation per se does not provide a more global safeguard against AID-mediated activity. These data also suggest that AID, and possibly other related cytidine Deaminases, might represent a more rapid alternative to bisulfite sequencing for identifying methylated-CpG motifs.
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activation induced cytidine Deaminase deaminates deoxycytidine on single stranded dna but requires the action of rnase
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Ronda Bransteitter, Phuong Pham, Matthew D. Scharff, Myron F GoodmanAbstract:The expression of activation-induced cytidine Deaminase (AID) is prerequisite to a “trifecta” of key molecular events in B cells: class-switch recombination and somatic hypermutation in humans and mice and gene conversion in chickens. Although this critically important enzyme shares common sequence motifs with apolipoprotein B mRNA-editing enzyme, and exhibits Deaminase activity on free deoxycytidine in solution, it has not been shown to act on either RNA or DNA. Recent mutagenesis data in Escherichia coli suggest that AID may deaminate dC on DNA, but its putative biochemical activities on either DNA or RNA remained a mystery. Here, we show that AID catalyzes deamination of dC residues on single-stranded DNA in vitro but not on double-stranded DNA, RNA–DNA hybrids, or RNA. Remarkably, it has no measurable Deaminase activity on single-stranded DNA unless pretreated with RNase to remove inhibitory RNA bound to AID. AID catalyzes dC → dU deamination activity most avidly on double-stranded DNA substrates containing a small “transcription-like” single-stranded DNA bubble, suggesting a targeting mechanism for this enigmatic enzyme during somatic hypermutation.
Michael S Neuberger - One of the best experts on this subject based on the ideXlab platform.
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dna deamination in immunity aid in the context of its apobec relatives
Advances in Immunology, 2007Co-Authors: Silvestro G Conticello, Marcandre Langlois, Zizhen Yang, Michael S NeubergerAbstract:Abstract The activation‐induced cytidine Deaminase (AID)/apolipoprotein B RNA‐editing catalytic component (APOBEC) family is a vertebrate‐restricted subgrouping of a superfamily of zinc (Zn)‐dependent Deaminases that has members distributed throughout the biological world. AID and APOBEC2 are the oldest family members with APOBEC1 and the APOBEC3s being later arrivals restricted to placental mammals. Many AID/APOBEC family members exhibit cytidine Deaminase activity on polynucleotides, although in different physiological contexts. Here, we examine the AID/APOBEC proteins in the context of the entire Zn‐dependent Deaminase superfamily. On the basis of secondary structure predictions, we propose that the cytosine and tRNA Deaminases are likely to provide better structural paradigms for the AID/APOBEC family than do the cytidine Deaminases, to which they have conventionally been compared. These comparisons yield predictions concerning likely polynucleotide‐interacting residues in AID/APOBEC3s, predictions that are supported by mutagenesis studies. We also focus on a specific comparison between AID and the APOBEC3s. Both are DNA Deaminases that function in immunity and are responsible for the hypermutation of their target substrates. AID functions in the adaptive immune system to diversify antibodies with targeted DNA deamination being central to this function. APOBEC3s function as part of an innate pathway of immunity to retroviruses with targeted DNA deamination being central to their activity in retroviral hypermutation. However, the mechanism by which the APOBEC3s fulfill their function of retroviral restriction remains unresolved.
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Mutational comparison of the single-domained APOBEC3C and double-domained APOBEC3F/G anti-retroviral cytidine Deaminases provides insight into their DNA target site specificities
Nucleic Acids Research, 2005Co-Authors: Marcandre Langlois, Silvestro G Conticello, Rupert Beale, Michael S NeubergerAbstract:Human APOBEC3F and APOBEC3G are double-domained Deaminases that can catalyze dC→dU deamination in HIV-1 and MLV retroviral DNA replication intermediates, targeting T–C or C–C dinucleotides, respectively. HIV-1 antagonizes their action through its vif gene product, which has been shown (at least in the case of APOBEC3G) to interact with the N-terminal domain of the Deaminase, triggering its degradation. Here, we compare APOBEC3F and APOBEC3G to APOBEC3C, a single-domained Deaminase that can also act on both HIV-1 and MLV. We find that whereas APOBEC3C contains all the information necessary for both Vif-binding and cytidine Deaminase activity in a single domain, it is the C-terminal domain of APOBEC3F and APOBEC3G that confer their target site specificity for cytidine deamination. We have exploited the fact that APOBEC3C, whilst highly homologous to the C-terminal domain of APOBEC3F, exhibits a distinct target site specificity (preferring Y–C dinucleotides) in order to identify residues in APOBEC3F that might affect its target site specificity. We find that this specificity can be altered by single amino acid substitutions at several distinct positions, suggesting that the strong dependence of APOBEC3-mediated deoxycytidine deamination on the 5′-flanking nucleotide is sensitive to relatively subtle changes in the APOBEC3 structure. The approach has allowed the isolation of APOBEC3 DNA mutators that exhibit novel target site preferences.
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comparison of the differential context dependence of dna deamination by apobec enzymes correlation with mutation spectra in vivo
Journal of Molecular Biology, 2004Co-Authors: Rupert Beale, Svend K Petersenmahrt, Reuben S Harris, Ian N Watt, Cristina Rada, Michael S NeubergerAbstract:To investigate the extent to which in vivo mutation spectra might reflect the intrinsic specificities of active mutators, genetic and biochemical assays were used to analyse the DNA target specificities of cytidine Deaminases of the APOBEC family. The results reveal the critical importance of nucleotides immediately 5′ of the targeted C for the specificity of all three enzymes studied (AID, APOBEC1 and APOBEC3G). At position −1, APOBEC1 showed a marked preference for dT, AID for dA/dG and APOBEC3G a strong preference for dC. Furthermore, AID and APOBEC3G showed distinct dependence on the nucleotide at position −2 with dA/dT being favoured by AID and dC by APOBEC3G. Most if not all activity of the recombinant Deaminases on free dC could be attributed to low-level contamination by host enzymes. The target preference of APOBEC3G supports it being a major but possibly not sole contributor to HIV hypermutation without making it a dominant contribution to general HIV sequence variation. The specificity of AID as deduced from the genetic assay (which relies on inactivation of sacB of Bacillus subtilis) agrees well with that deduced by Pham et al. using an in vitro assay although we postulate that major intrinsic mutational hotspots in immunoglobulin V genes in vivo might reflect favoured sites of AID action being generated by proximal DNA targets located on opposite DNA strands. The target specificity of AID also accords with the spectrum of mutations observed in B lymphoma-associated oncogenes. The possibility of Deaminase involvement in non-lymphoid human tumours is hinted at by tissue-specific differences in the spectra of dC transitions in tumour-suppressor genes. Thus, the patterns of hypermutation in antibodies and retroviruses owe much to the intrinsic sequence preferences of the AID/APOBEC family of DNA Deaminases: analogous biases might also contribute to the spectra of cancer-associated mutation.
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in vitro deamination of cytosine to uracil in single stranded dna by apolipoprotein b editing complex catalytic subunit 1 apobec1
Journal of Biological Chemistry, 2003Co-Authors: Svend K Petersenmahrt, Michael S NeubergerAbstract:Abstract Apolipoprotein B-editing complex catalytic subunit 1 (APOBEC1) is the catalytic component of an RNA-editing complex that deaminates C6666 → U in apolipoprotein B RNA in gastrointestinal tissue, thereby generating a premature stop codon. Whereas RNA is the physiological substrate of APOBEC1, recent experiments have strongly indicated that, when expressed in bacteria, APOBEC1 and some of its homologues can deaminate cytosine in DNA. Indeed, genetic evidence demonstrates that the physiological function of activation-induced Deaminase, a B lymphocyte-specific APOBEC1 homologue, is to perform targeted deamination of cytosine within the immunoglobulin locus, thereby triggering antibody gene diversification. However, biochemical evidence of in vitro DNA deamination by members of the APOBEC family is still needed. Here, we show that deamination of cytosine to uracil in DNA can be achieved in vitro using partially purified APOBEC1 from extracts of transformed Escherichia coli. Thus, APOBEC1 can deaminate cytosine in both RNA and DNA. Strikingly, its activity on DNA is specific for single-stranded DNA and exhibits dependence on local sequence context.
Hiroshi Matsuo - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity
Nature Communications, 2017Co-Authors: Takahide Kouno, Tania V. Silvas, Brendan J. Hilbert, Shivender M. D. Shandilya, Markus F. Bohn, Brian A. Kelch, William E. Royer, Mohan Somasundaran, Nese Kurt Yilmaz, Hiroshi MatsuoAbstract:Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine Deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine Deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A–ssDNA complex defines the 5′–3′ directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide Deaminases, thereby opening the door for the design of mechanistic-based therapeutics. Cytidine Deaminases are evolutionarily conserved enzymes that edit genomes by deaminating cytidine to uridine. Here the authors present the crystal structure of APOBEC3A with a single-stranded DNA substrate bound in the active site to shed light on the mechanism and specificity of substrate recognition.
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structure of the dna Deaminase domain of the hiv 1 restriction factor apobec3g
Nature, 2008Co-Authors: Kuan Ming Chen, Reuben S Harris, Elena Harjes, Phillip J Gross, Amr Fahmy, Yongjian Lu, Keisuke Shindo, Hiroshi MatsuoAbstract:The cellular cytosine Deaminase APOBEC3G inhibits replication of HIV lacking the viral protein Vif; here the NMR structure of a mutant form of the catalytic domain of APOBEC3G is reported, and a model for the APOBEC–DNA interaction is proposed. The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA Deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons1,2,3,4,5,6. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif7. APOBEC3G is a member of a family of polynucleotide cytosine Deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA8,9,10. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine Deaminase or anti-viral activity11,12,13,14,15,16. Here we report a solution structure of the human APOBEC3G catalytic domain. Five α-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five β-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.
Vitor Hugo Balasco Serrão - One of the best experts on this subject based on the ideXlab platform.
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spectroscopic and calorimetric assays reveal dependence on dctp and two metals zn2 mg2 for enzymatic activity of schistosoma mansoni deoxycytidylate dcmp Deaminase
Biochimica et Biophysica Acta, 2017Co-Authors: Jessica Fernandes Scortecci, J Cheleski, J R Torini, Larissa Romanello, Vitor Hugo Balasco Serrão, Ricardo Demarco, Humberto Dmuniz PereiraAbstract:Abstract The parasite Schistosoma mansoni possess all pathways for pyrimidine biosynthesis, whereby Deaminases play an essential role in the thymidylate cycle, a crucial step to controlling the ratio between cytidine and uridine nucleotides. In this study, we heterologously expressed and purified the deoxycytidylate (dCMP) Deaminase from S. mansoni to obtain structural, biochemical and kinetic information. Small-angle X-ray scattering of this enzyme showed that it is organized as a hexamer in solution. Isothermal titration calorimetry was used to determine the kinetic constants for dCMP-dUMP conversion and the role of dCTP and dTTP in enzymatic regulation. We evaluated the metals involved in activating the enzyme and show for the first time the dependence of correct folding on the interaction of two metals. This study provides information that may be useful for understanding the regulatory mechanisms involved in the metabolic pathways of S. mansoni. Thus, improving our understanding of the function of these essential pathways for parasite metabolism and showing for the first time the hitherto unknown Deaminase function in this parasite.
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Spectroscopic and calorimetric assays reveal dependence on dCTP and two metals (Zn2++Mg2+) for enzymatic activity of Schistosoma mansoni deoxycytidylate (dCMP) Deaminase.
Biochimica et biophysica acta. Proteins and proteomics, 2017Co-Authors: Jessica Fernandes Scortecci, J Cheleski, J R Torini, Larissa Romanello, Vitor Hugo Balasco Serrão, Ricardo Demarco, Humberto D'muniz PereiraAbstract:The parasite Schistosoma mansoni possess all pathways for pyrimidine biosynthesis, whereby Deaminases play an essential role in the thymidylate cycle, a crucial step to controlling the ratio between cytidine and uridine nucleotides. In this study, we heterologously expressed and purified the deoxycytidylate (dCMP) Deaminase from S. mansoni to obtain structural, biochemical and kinetic information. Small-angle X-ray scattering of this enzyme showed that it is organized as a hexamer in solution. Isothermal titration calorimetry was used to determine the kinetic constants for dCMP-dUMP conversion and the role of dCTP and dTTP in enzymatic regulation. We evaluated the metals involved in activating the enzyme and show for the first time the dependence of correct folding on the interaction of two metals. This study provides information that may be useful for understanding the regulatory mechanisms involved in the metabolic pathways of S. mansoni. Thus, improving our understanding of the function of these essential pathways for parasite metabolism and showing for the first time the hitherto unknown Deaminase function in this parasite.
Jessica Fernandes Scortecci - One of the best experts on this subject based on the ideXlab platform.
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spectroscopic and calorimetric assays reveal dependence on dctp and two metals zn2 mg2 for enzymatic activity of schistosoma mansoni deoxycytidylate dcmp Deaminase
Biochimica et Biophysica Acta, 2017Co-Authors: Jessica Fernandes Scortecci, J Cheleski, J R Torini, Larissa Romanello, Vitor Hugo Balasco Serrão, Ricardo Demarco, Humberto Dmuniz PereiraAbstract:Abstract The parasite Schistosoma mansoni possess all pathways for pyrimidine biosynthesis, whereby Deaminases play an essential role in the thymidylate cycle, a crucial step to controlling the ratio between cytidine and uridine nucleotides. In this study, we heterologously expressed and purified the deoxycytidylate (dCMP) Deaminase from S. mansoni to obtain structural, biochemical and kinetic information. Small-angle X-ray scattering of this enzyme showed that it is organized as a hexamer in solution. Isothermal titration calorimetry was used to determine the kinetic constants for dCMP-dUMP conversion and the role of dCTP and dTTP in enzymatic regulation. We evaluated the metals involved in activating the enzyme and show for the first time the dependence of correct folding on the interaction of two metals. This study provides information that may be useful for understanding the regulatory mechanisms involved in the metabolic pathways of S. mansoni. Thus, improving our understanding of the function of these essential pathways for parasite metabolism and showing for the first time the hitherto unknown Deaminase function in this parasite.
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Spectroscopic and calorimetric assays reveal dependence on dCTP and two metals (Zn2++Mg2+) for enzymatic activity of Schistosoma mansoni deoxycytidylate (dCMP) Deaminase.
Biochimica et biophysica acta. Proteins and proteomics, 2017Co-Authors: Jessica Fernandes Scortecci, J Cheleski, J R Torini, Larissa Romanello, Vitor Hugo Balasco Serrão, Ricardo Demarco, Humberto D'muniz PereiraAbstract:The parasite Schistosoma mansoni possess all pathways for pyrimidine biosynthesis, whereby Deaminases play an essential role in the thymidylate cycle, a crucial step to controlling the ratio between cytidine and uridine nucleotides. In this study, we heterologously expressed and purified the deoxycytidylate (dCMP) Deaminase from S. mansoni to obtain structural, biochemical and kinetic information. Small-angle X-ray scattering of this enzyme showed that it is organized as a hexamer in solution. Isothermal titration calorimetry was used to determine the kinetic constants for dCMP-dUMP conversion and the role of dCTP and dTTP in enzymatic regulation. We evaluated the metals involved in activating the enzyme and show for the first time the dependence of correct folding on the interaction of two metals. This study provides information that may be useful for understanding the regulatory mechanisms involved in the metabolic pathways of S. mansoni. Thus, improving our understanding of the function of these essential pathways for parasite metabolism and showing for the first time the hitherto unknown Deaminase function in this parasite.
