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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 rna polymerase ii transcription dependent Deamination of igv dna
Nucleic Acids Research, 2019Co-Authors: Phuong Pham, Sohail Malik, Chiho Mak, Peter C Calabrese, Robert G Roeder, Myron F GoodmanAbstract:Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes to produce high-affinity antibodies. SHM requires IgV transcription by RNA polymerase II (Pol II). A eukaryotic transcription system including AID has not been reported previously. Here, we reconstitute AID-catalyzed Deamination during Pol II transcription elongation in conjunction with DSIF transcription factor. C→T mutations occur at similar frequencies on non-transcribed strand (NTS) and transcribed strand (TS) DNA. In contrast, bacteriophage T7 Pol generates NTS mutations predominantly. AID-Pol II mutations are strongly favored in WRC and WGCW overlapping hot motifs (W = A or T, R = A or G) on both DNA strands. Single mutations occur on 70% of transcribed DNA clones. Mutations are correlated over a 15 nt distance in multiply mutated clones, suggesting that Deaminations are catalyzed processively within a stalled or backtracked transcription bubble. Site-by-site comparisons for biochemical and human memory B-cell mutational spectra in an IGHV3-23*01 target show strongly favored Deaminations occurring in the antigen-binding complementarity determining regions (CDR) compared to the framework regions (FW). By exhibiting consistency with B-cell SHM, our in vitro data suggest that biochemically defined reconstituted Pol II transcription systems can be used to investigate how, when and where AID is targeted.
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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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structural model for deoxycytidine Deamination mechanisms of the hiv 1 inactivation enzyme apobec3g
Journal of Biological Chemistry, 2010Co-Authors: Linda Chelico, Courtney Prochnow, Dorothy A Erie, Xiaojiang S Chen, Myron F GoodmanAbstract:Abstract APOBEC3G (Apo3G) is a single-stranded DNA-dependent deoxycytidine deaminase, which, in the absence of the human immunodeficiency virus (HIV) viral infectivity factor, is encapsulated into HIV virions. Subsequently, Apo3G triggers viral inactivation by processively deaminating C→U, with 3′→5′ polarity, on nascent minus-strand cDNA. Apo3G has a catalytically inactive N-terminal CD1 domain and an active C-terminal CD2 domain. Apo3G exists as monomers, dimers, tetramers, and higher order oligomers whose distributions depend on DNA substrate and salt. Here we use multiangle light scattering and atomic force microscopy to identify oligomerization states of Apo3G. A double mutant (F126A/W127A), designed to disrupt dimerization at the predicted CD1-CD1 dimer interface, predominantly converts Apo3G to a monomer that binds single-stranded DNA, Alu RNA, and catalyzes processive C→U Deaminations with 3′→5′ Deamination polarity, similar to native Apo3G. The CD1 domain is essential for both processivity and polarity. We propose a structure-based model to explain the scanning and catalytic behavior of Apo3G.
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impact of phosphorylation and phosphorylation null mutants on the activity and Deamination specificity of activation induced cytidine deaminase
Journal of Biological Chemistry, 2008Co-Authors: Phuong Pham, Marcus B Smolka, Peter Calabrese, Alice Landolph, Ke Zhang, Huilin Zhou, Myron F GoodmanAbstract:Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination in B cells by deaminating C → U on transcribed DNA. Here we analyze the role of phosphorylation and phosphorylation-null mutants on the biochemical behavior of AID, including enzyme specific activity, processivity, Deamination spectra, Deamination motif specificity, and transcription-dependent Deamination in the presence and absence of RPA. We show that a small fraction of recombinant human AID expressed in Sf9 insect cells is phosphorylated at previously identified residues Ser38 and Thr27 and also at Ser41 and Ser43. S43P AID has been identified in a patient with hyper-IgM immunodeficiency syndrome. Ser-substituted phosphorylation-null mutants (S38A, S41A, S43A, and S43P) exhibit wild type (WT) activity on single-stranded DNA. Deamination of transcribed double-stranded DNA is similar for WT and mutant AID and occurs with or without RPA. Although WT and AID mutants catalyze processive Deamination favoring canonical WRC hot spot motifs (where W represents A/T and R is A/G), their Deamination spectra differ significantly. The differences between the WT and AID mutants appear to be caused by the replacement of Ser as opposed to an absence of phosphorylation. The spectral differences reflect a marked change in Deamination efficiencies in two motifs, GGC and AGC, which are preferred by mutant AID but disfavored by WT AID. Both motifs occur with exceptionally high frequency in human switch regions, suggesting a possible relationship between AID Deamination specificity and a loss of antibody diversification.
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processive aid catalysed cytosine Deamination on single stranded dna simulates somatic hypermutation
Nature, 2003Co-Authors: Phuong Pham, Ronda Bransteitter, John Petruska, Myron F GoodmanAbstract:Activation-induced cytidine deaminase (AID) is a protein required for B cells to undergo class switch recombination and somatic hypermutation (SHM)—two processes essential for producing high-affinity antibodies1. Purified AID catalyses the Deamination of C to U on single-stranded (ss)DNA2,3,4. Here, we show in vitro that AID-catalysed C Deaminations occur preferentially on 5′ WRC sequences in accord with SHM spectra observed in vivo. Although about 98% of DNA clones suffer no mutations, most of the remaining mutated clones have 10–70 C to T transitions per clone. Therefore, AID carries out multiple C Deaminations on individual DNA strands, rather than jumping from one strand to another. The avid binding of AID to ssDNA could result from its large net positive charge (+11) at pH 7.0, owing to a basic amino-terminal domain enriched in arginine and lysine. Furthermore, AID exhibits a 15-fold preference for C Deamination on the non-transcribed DNA strand exposed by RNA polymerase than the transcribed strand protected as a RNA–DNA hybrid. These Deamination results on ssDNA bear relevance to three characteristic features of SHM: preferential mutation at C sites within WRC hotspot sequences, the broad clonal mutagenic heterogeneity of antibody variable regions targeted for mutation5,6, and the requirement for active transcription to obtain mutagenesis7,8.
Phuong Pham - One of the best experts on this subject based on the ideXlab platform.
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aid rna polymerase ii transcription dependent Deamination of igv dna
Nucleic Acids Research, 2019Co-Authors: Phuong Pham, Sohail Malik, Chiho Mak, Peter C Calabrese, Robert G Roeder, Myron F GoodmanAbstract:Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes to produce high-affinity antibodies. SHM requires IgV transcription by RNA polymerase II (Pol II). A eukaryotic transcription system including AID has not been reported previously. Here, we reconstitute AID-catalyzed Deamination during Pol II transcription elongation in conjunction with DSIF transcription factor. C→T mutations occur at similar frequencies on non-transcribed strand (NTS) and transcribed strand (TS) DNA. In contrast, bacteriophage T7 Pol generates NTS mutations predominantly. AID-Pol II mutations are strongly favored in WRC and WGCW overlapping hot motifs (W = A or T, R = A or G) on both DNA strands. Single mutations occur on 70% of transcribed DNA clones. Mutations are correlated over a 15 nt distance in multiply mutated clones, suggesting that Deaminations are catalyzed processively within a stalled or backtracked transcription bubble. Site-by-site comparisons for biochemical and human memory B-cell mutational spectra in an IGHV3-23*01 target show strongly favored Deaminations occurring in the antigen-binding complementarity determining regions (CDR) compared to the framework regions (FW). By exhibiting consistency with B-cell SHM, our in vitro data suggest that biochemically defined reconstituted Pol II transcription systems can be used to investigate how, when and where AID is targeted.
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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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impact of phosphorylation and phosphorylation null mutants on the activity and Deamination specificity of activation induced cytidine deaminase
Journal of Biological Chemistry, 2008Co-Authors: Phuong Pham, Marcus B Smolka, Peter Calabrese, Alice Landolph, Ke Zhang, Huilin Zhou, Myron F GoodmanAbstract:Activation-induced cytidine deaminase (AID) initiates somatic hypermutation and class switch recombination in B cells by deaminating C → U on transcribed DNA. Here we analyze the role of phosphorylation and phosphorylation-null mutants on the biochemical behavior of AID, including enzyme specific activity, processivity, Deamination spectra, Deamination motif specificity, and transcription-dependent Deamination in the presence and absence of RPA. We show that a small fraction of recombinant human AID expressed in Sf9 insect cells is phosphorylated at previously identified residues Ser38 and Thr27 and also at Ser41 and Ser43. S43P AID has been identified in a patient with hyper-IgM immunodeficiency syndrome. Ser-substituted phosphorylation-null mutants (S38A, S41A, S43A, and S43P) exhibit wild type (WT) activity on single-stranded DNA. Deamination of transcribed double-stranded DNA is similar for WT and mutant AID and occurs with or without RPA. Although WT and AID mutants catalyze processive Deamination favoring canonical WRC hot spot motifs (where W represents A/T and R is A/G), their Deamination spectra differ significantly. The differences between the WT and AID mutants appear to be caused by the replacement of Ser as opposed to an absence of phosphorylation. The spectral differences reflect a marked change in Deamination efficiencies in two motifs, GGC and AGC, which are preferred by mutant AID but disfavored by WT AID. Both motifs occur with exceptionally high frequency in human switch regions, suggesting a possible relationship between AID Deamination specificity and a loss of antibody diversification.
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processive aid catalysed cytosine Deamination on single stranded dna simulates somatic hypermutation
Nature, 2003Co-Authors: Phuong Pham, Ronda Bransteitter, John Petruska, Myron F GoodmanAbstract:Activation-induced cytidine deaminase (AID) is a protein required for B cells to undergo class switch recombination and somatic hypermutation (SHM)—two processes essential for producing high-affinity antibodies1. Purified AID catalyses the Deamination of C to U on single-stranded (ss)DNA2,3,4. Here, we show in vitro that AID-catalysed C Deaminations occur preferentially on 5′ WRC sequences in accord with SHM spectra observed in vivo. Although about 98% of DNA clones suffer no mutations, most of the remaining mutated clones have 10–70 C to T transitions per clone. Therefore, AID carries out multiple C Deaminations on individual DNA strands, rather than jumping from one strand to another. The avid binding of AID to ssDNA could result from its large net positive charge (+11) at pH 7.0, owing to a basic amino-terminal domain enriched in arginine and lysine. Furthermore, AID exhibits a 15-fold preference for C Deamination on the non-transcribed DNA strand exposed by RNA polymerase than the transcribed strand protected as a RNA–DNA hybrid. These Deamination results on ssDNA bear relevance to three characteristic features of SHM: preferential mutation at C sites within WRC hotspot sequences, the broad clonal mutagenic heterogeneity of antibody variable regions targeted for mutation5,6, and the requirement for active transcription to obtain mutagenesis7,8.
Susan R Ross - One of the best experts on this subject based on the ideXlab platform.
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apobec3 inhibition of mouse mammary tumor virus infection the role of cytidine Deamination versus inhibition of reverse transcription
Journal of Virology, 2013Co-Authors: Alyssa L Macmillan, Rahul M Kohli, Susan R RossAbstract:ABSTRACT The apolipoprotein B editing complex 3 (APOBEC3) family of proteins is a group of intrinsic antiviral factors active against a number of retroviral pathogens, including HIV in humans and mouse mammary tumor virus (MMTV) in mice. APOBEC3 restricts its viral targets through cytidine Deamination of viral DNA during reverse transcription or via deaminase-independent means. Here, we used virions from the mammary tissue of MMTV-infected inbred wild-type mice with different allelic APOBEC3 variants (APOBEC3 BALB and APOBEC3 BL/6 ) and knockout mice to determine whether cytidine Deamination was important for APOBEC39s anti-MMTV activity. First, using anti-murine APOBEC3 antiserum, we showed that both APOBEC3 allelic variants are packaged into the cores of milk-borne virions produced in vivo . Next, using an in vitro Deamination assay, we determined that virion-packaged APOBEC3 retains its Deamination activity and that allelic differences in APOBEC3 affect the sequence specificity. In spite of this in vitro activity, cytidine Deamination by virion-packaged APOBEC3 of MMTV early reverse transcription DNA occurred only at low levels. Instead, the major means by which in vivo virion-packaged APOBEC3 restricted virus was through inhibition of early reverse transcription in both cell-free virions and in vitro infection assays. Moreover, the different wild-type alleles varied in their ability to inhibit this step. Our data suggest that while APOBEC3-mediated cytidine Deamination of MMTV may occur, it is not the major means by which APOBEC3 restricts MMTV infection in vivo . This may reflect the long-term coexistence of MMTV and APOBEC3 in mice.
Linda Chelico - One of the best experts on this subject based on the ideXlab platform.
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intensity of deoxycytidine Deamination of hiv 1 proviral dna by the retroviral restriction factor apobec3g is mediated by the noncatalytic domain
Journal of Biological Chemistry, 2011Co-Authors: Yuqing Feng, Linda ChelicoAbstract:Abstract APOBEC3G is a single-stranded (ss) DNA deaminase that restricts replication of HIV-1 by inducing viral genome mutagenesis through Deamination of cytosine to uracil on HIV-1 cDNA. APOBEC3G has polydisperse oligomeric states and deaminates ssDNA processively through jumping and sliding. APOBEC3G has a catalytically inactive N-terminal CD1 domain that mediates processivity and an active C-terminal CD2 domain that catalyzes Deaminations. Here, we assess the determinants of APOBEC3G Deamination efficiency mediated by the CD1 domain by comparing native APOBEC3G and two CD1 mutants, a monomeric mutant (F126A/W127A) and a clinical mutant associated with high viral loads (H186R). Biochemical assays on ssDNA or partially dsDNA and with a reconstituted HIV replication system demonstrate that both mutants of Apo3G have altered DNA scanning properties in either jumping (F126A/W127A) or sliding (H186R), which results in decreased abilities to induce mutagenesis during reverse transcription. The data reveal a functionality for Apo3G oligomers in Deamination and provide the first biochemical characterization of the clinical mutant H186R. The data demonstrate that the balance between the jumping and sliding of Apo3G is needed for efficient mutational inactivation of HIV-1.
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structural model for deoxycytidine Deamination mechanisms of the hiv 1 inactivation enzyme apobec3g
Journal of Biological Chemistry, 2010Co-Authors: Linda Chelico, Courtney Prochnow, Dorothy A Erie, Xiaojiang S Chen, Myron F GoodmanAbstract:Abstract APOBEC3G (Apo3G) is a single-stranded DNA-dependent deoxycytidine deaminase, which, in the absence of the human immunodeficiency virus (HIV) viral infectivity factor, is encapsulated into HIV virions. Subsequently, Apo3G triggers viral inactivation by processively deaminating C→U, with 3′→5′ polarity, on nascent minus-strand cDNA. Apo3G has a catalytically inactive N-terminal CD1 domain and an active C-terminal CD2 domain. Apo3G exists as monomers, dimers, tetramers, and higher order oligomers whose distributions depend on DNA substrate and salt. Here we use multiangle light scattering and atomic force microscopy to identify oligomerization states of Apo3G. A double mutant (F126A/W127A), designed to disrupt dimerization at the predicted CD1-CD1 dimer interface, predominantly converts Apo3G to a monomer that binds single-stranded DNA, Alu RNA, and catalyzes processive C→U Deaminations with 3′→5′ Deamination polarity, similar to native Apo3G. The CD1 domain is essential for both processivity and polarity. We propose a structure-based model to explain the scanning and catalytic behavior of Apo3G.
Rahul M Kohli - One of the best experts on this subject based on the ideXlab platform.
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apobec3 inhibition of mouse mammary tumor virus infection the role of cytidine Deamination versus inhibition of reverse transcription
Journal of Virology, 2013Co-Authors: Alyssa L Macmillan, Rahul M Kohli, Susan R RossAbstract:ABSTRACT The apolipoprotein B editing complex 3 (APOBEC3) family of proteins is a group of intrinsic antiviral factors active against a number of retroviral pathogens, including HIV in humans and mouse mammary tumor virus (MMTV) in mice. APOBEC3 restricts its viral targets through cytidine Deamination of viral DNA during reverse transcription or via deaminase-independent means. Here, we used virions from the mammary tissue of MMTV-infected inbred wild-type mice with different allelic APOBEC3 variants (APOBEC3 BALB and APOBEC3 BL/6 ) and knockout mice to determine whether cytidine Deamination was important for APOBEC39s anti-MMTV activity. First, using anti-murine APOBEC3 antiserum, we showed that both APOBEC3 allelic variants are packaged into the cores of milk-borne virions produced in vivo . Next, using an in vitro Deamination assay, we determined that virion-packaged APOBEC3 retains its Deamination activity and that allelic differences in APOBEC3 affect the sequence specificity. In spite of this in vitro activity, cytidine Deamination by virion-packaged APOBEC3 of MMTV early reverse transcription DNA occurred only at low levels. Instead, the major means by which in vivo virion-packaged APOBEC3 restricted virus was through inhibition of early reverse transcription in both cell-free virions and in vitro infection assays. Moreover, the different wild-type alleles varied in their ability to inhibit this step. Our data suggest that while APOBEC3-mediated cytidine Deamination of MMTV may occur, it is not the major means by which APOBEC3 restricts MMTV infection in vivo . This may reflect the long-term coexistence of MMTV and APOBEC3 in mice.
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AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation
Nature Chemical Biology, 2012Co-Authors: Christopher S Nabel, Hana L Goldschmidt, Li Shen, James T Stivers, Yu Ye, Yi Zhang, Rahul M KohliAbstract:AID/APOBEC deaminases, which convert cytosine bases to uracils in DNA and RNA, have recently been assigned a role in epigenetic regulation as components of DNA demethylation pathways. A systematic study shows that AID/APOBEC enzymes preferentially deaminate unmodified cytosine over its C5-modified forms, calling into question the plausibility of deaminase-mediated DNA demethylation pathways. Activation-induced deaminase (AID)/APOBEC–family cytosine deaminases, known to function in diverse cellular processes from antibody diversification to mRNA editing, have also been implicated in DNA demethylation, a major process for transcriptional activation. Although oxidation-dependent pathways for demethylation have been described, pathways involving Deamination of either 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC) have emerged as alternatives. Here we address the biochemical plausibility of Deamination-coupled demethylation. We found that purified AID/APOBECs have substantially reduced activity on 5mC relative to cytosine, their canonical substrate, and no detectable Deamination of 5hmC. This finding was explained by the reactivity of a series of modified substrates, where steric bulk was increasingly detrimental to Deamination. Further, upon AID/APOBEC overexpression, the Deamination product of 5hmC was undetectable in genomic DNA, whereas oxidation intermediates remained detectable. Our results indicate that the steric requirements for cytosine Deamination are one intrinsic barrier to the proposed function of deaminases in DNA demethylation.
