Dissociation of in vitro DNA deamination activity and physiological functions of AID mutants.
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ABSTRACT:
SUBMITTER: Shivarov V
PROVIDER: S-EPMC2572969 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The AID/APOBEC polynucleotide cytidine deaminases have historically been classified as either DNA mutators or RNA editors based on their first identified nucleic acid substrate preference. DNA mutators can generate functional diversity at antibody genes but also cause genomic instability in cancer. RNA editors can generate informational diversity in the transcriptome of innate immune cells, and of cancer cells. Members of both classes can act as antiviral restriction factors. Recent structural work has illuminated differences and similarities between AID/APOBEC enzymes that can catalyse DNA mutation, RNA editing or both, suggesting that the strict functional classification of members of this family should be reconsidered. As many of these enzymes have been employed for targeted genome (or transcriptome) editing, a more holistic understanding will help improve the design of therapeutically relevant programmable base editors.
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Project description: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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Project description: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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Project description:The AID/APOBEC family of enzymes in higher vertebrates converts cytosines in DNA or RNA to uracil. They play a role in antibody maturation and innate immunity against viruses, and have also been implicated in the demethylation of DNA during early embryogenesis. This is based in part on reported ability of activation-induced deaminase (AID) to deaminate 5-methylcytosines (5mC) to thymine. We have reexamined this possibility for AID and two members of human APOBEC3 family using a novel genetic system in Escherichia coli. Our results show that while all three genes show strong ability to convert C to U, only APOBEC3A is an efficient deaminator of 5mC. To confirm this, APOBEC3A was purified partially and used in an in vitro deamination assay. We found that APOBEC3A can deaminate 5mC efficiently and this activity is comparable to its C to U deamination activity. When the DNA-binding segment of AID was replaced with the corresponding segment from APOBEC3A, the resulting hybrid had much higher ability to convert 5mC to T in the genetic assay. These and other results suggest that the human AID deaminates 5mC's only weakly because the 5-methyl group fits poorly in its DNA-binding pocket.
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Project description: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 Ser(38) and Thr(27) and also at Ser(41) and Ser(43). 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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Project description:Mitochondrial DNA helicase, also called Twinkle, is essential for mtDNA maintenance. Its helicase domain shares high homology with helicases from superfamily 4. Structural analyses of helicases from this family indicate that carboxyl-terminal residues contribute to NTP hydrolysis required for translocation and DNA unwinding, yet genetic and biochemical information is very limited. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying a series of deletion and alanine substitution mutations in the carboxyl terminus and identify critical residues between amino acids 572 and 596 of the 613 amino acid polypeptide that are essential for mitochondrial DNA helicase function in vivo. Likewise, amino acid substitution mutants K574A, R576A, Y577A, F588A, and F595A show dose-dependent dominant-negative phenotypes. Arg-576 and Phe-588 are analogous to the arginine finger and base stack of other helicases, including the bacteriophage T7 gene 4 protein and bacterial DnaB helicase, respectively. We show here that representative human recombinant proteins that are analogous to the alanine substitution mutants exhibit defects in nucleotide hydrolysis. Our findings may be applicable to understand the role of the carboxyl-terminal region in superfamily 4 DNA helicases in general.
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Project description:APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like) is a family of enzymes that deaminates cytosine (C) to uracil (U) on nucleic acid. APOBEC3B (A3B) functions in innate immunity against intrinsic and invading retroelements and viruses. A3B can also induce genomic DNA mutations to cause cancer. A3B contains two cytosine deaminase domains (CD1, CD2), and there are conflicting reports about whether both domains are active. Here we demonstrate that only CD2 of A3B (A3BCD2) has C deamination activity. We also reveal that both A3B and A3BCD2 can deaminate methylcytosine (mC). Guided by structural and functional analysis, we successfully engineered A3BCD2 to gain over two orders of magnitude higher activity for mC deamination. Important determinants that contribute to the activity and selectivity for mC deamination have been identified, which reveals that multiple elements, rather than single ones, contribute to the mC deamination activity and selectivity in A3BCD2 and possibly other APOBECs.
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Project description:Cohesin is a member of the Smc family of protein complexes that mediates higher-order chromosome structure by tethering different regions of chromatin. We present a new in vitro system that assembles cohesin-DNA complexes with in vivo properties. The assembly of these physiological salt-resistant complexes requires the cohesin holo-complex, its ability to bind ATP, the cohesin loader Scc2p and a closed DNA topology. Both the number of cohesin molecules bound to the DNA substrate and their distribution on the DNA substrate are limited. Cohesin and Scc2p bind preferentially to cohesin associated regions (CARs), DNA sequences with enriched cohesin binding in vivo. A subsequence of CARC1 promotes cohesin binding to neighboring sequences within CARC1. The enhancer-like function of this sequence is validated by in vivo deletion analysis. By demonstrating the physiological relevance of these in vitro assembled cohesin-DNA complexes, we establish our in vitro system as a powerful tool to elucidate the mechanism of cohesin and other Smc complexes.
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