Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNA interference (RNAi) is an extremely powerful and widely used gene silencing approach for reverse functional genomics and molecular therapeutics. In mammals, the conserved poly(ADP-ribose) polymerase 2 (PARP-2)/RNase P bidirectional control promoter simultaneously expresses both the PARP-2 protein and RNase P RNA by RNA polymerase II- and III-dependent mechanisms, respectively. To explore this unique bidirectional control system in RNAi-mediated gene silencing strategy, we have constructed two novel bidirectional expression vectors, pbiHsH1 and pbiMmH1, which contained the PARP-2/RNase P bidirectional control promoters from human and mouse, for simultaneous expression of both the protein-coding genes and short hairpin RNAs. Analyses of the dual transcriptional activities indicated that these two bidirectional expression vectors could not only express enhanced green fluorescent protein as a functional reporter but also simultaneously transcribe shLuc for inhibiting the firefly luciferase expression. In addition, to extend its utility for the establishment of inherited stable clones, we have also reconstructed this bidirectional expression system with the blasticidin S deaminase gene, an effective dominant drug resistance selectable marker, and examined both the selection and inhibition efficiencies in drug resistance and gene expression. Moreover, we have further demonstrated that this bidirectional expression system could efficiently co-regulate the functionally important genes, such as overexpression of tumor suppressor protein p53 and inhibition of anti-apoptotic protein Bcl-2 at the same time. In summary, the bidirectional expression vectors, pbiHsH1 and pbiMmH1, should provide a simple, convenient, and efficient novel tool for manipulating the gene function in mammalian cells.
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PMID:A novel bidirectional expression system for simultaneous expression of both the protein-coding genes and short hairpin RNAs in mammalian cells. 1633 9

The DNA sequence data of the somatic hypermutation (SHM) field published since 1984 has been critically reviewed. The analysis has revealed three strand biased mutation signatures. The first concerns the mutations generated at G:C base pairs in mice genetically deficient in uracil-DNA glycosylase and MSH2-MSH6-mediated mismatch repair. Such mice display the AID deaminase footprint and here C mutations exceed G mutations at least 1.5-fold. This supports earlier and more recent studies claiming that dC-to-dU deaminations occur preferentially in the single stranded DNA regions of the displaced nontranscribed strand (NTS) during transcription. The second concerns the signature generated in immunised mice where G mutations exceed C mutations by at least 1.7-fold. This is a newly identified strand bias which has previously gone undetected. It is consistent with the polynucleotide polymerisation signature of RNA polymerase II copying the template DNA strand carrying AID-mediated lesions generated at C bases, viz. uracils and abasic sites. A reverse transcription step would then need to intervene to fix the mutation pattern in DNA. The third concerns the long recognised strand biased signature generated in normal aged or actively immunised mice whereby A mutations exceed T mutations by two- to three-fold. It is argued that this pattern is best understood as a combination of adenosine-to-inosine (A-to-I) RNA editing followed by a reverse transcription step fixing the A-to-G, as well as A-to-T and A-to-C, as strand biased mutation signatures in DNA. The reasons why the AID-linked RNA polymerase II mutation signature had previously gone undetected are discussed with regard to limitations of standard PCR-based SHM assay techniques. It is concluded that the most economical SHM mechanism involves both DNA and RNA deaminations coupled to a reverse transcription process, most likely involving DNA polymerase eta acting in its reverse transcriptase mode. Experimental approaches to differentiate this RNA-based model from the standard DNA deamination model are discussed.
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PMID:Mechanism of somatic hypermutation: critical analysis of strand biased mutation signatures at A:T and G:C base pairs. 1906 97

Activation induced deaminase (AID) is globally targeted to immunoglobulin loci, preferentially focused to switch (S) regions and variable (V) regions, and prone to attack hotspot motifs. Nevertheless, AID deamination is not exclusive to Ig loci and the rules regulating AID targeting remain unclear. Transcription is critically required for class switch recombination and somatic hypermutation. Here, I consider the unique features associated with S region transcription leading to RNA polymerase II pausing, that in turn promote the introduction of activating chromatin remodeling, histone modifications and recruitment of AID to targeted S regions. These findings allow for a better understanding of the interplay between transcription, AID targeting and mistargeting to Ig and non-Ig loci.
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PMID:AID targeting is dependent on RNA polymerase II pausing. 2278 81

Immunoglobulin (Ig) class switch recombination (CSR) is responsible for diversification of antibody effector function during an immune response. This region-specific recombination event, between repetitive switch (S) DNA elements, is unique to B lymphocytes and is induced by activationinduced deaminase (AID). CSR is critically dependent on transcription of noncoding RNAs across S regions. However, mechanistic insight regarding this process has remained unclear. New studies indicate that long-range intrachromosomal interactions among IgH transcriptional elements organize the formation of the S/S synaptosome, as a prerequisite for CSR. This three-dimensional chromatin architecture simultaneously brings promoters and enhancers into close proximity to facilitate transcription. Here, we recount how transcription across S DNA promotes accumulation of RNA polymerase II, leading to the introduction of activating chromatin modifications and hyperaccessible chromatin that is amenable to AID activity.
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PMID:Three-dimensional architecture of the IgH locus facilitates class switch recombination. 2295 21

Antibody diversification requires the DNA deaminase AID to induce DNA instability at immunoglobulin (Ig) loci upon B cell stimulation. For efficient cytosine deamination, AID requires single-stranded DNA and needs to gain access to Ig loci, with RNA pol II transcription possibly providing both aspects. To understand these mechanisms, we isolated and characterized endogenous AID-containing protein complexes from the chromatin of diversifying B cells. The majority of proteins associated with AID belonged to RNA polymerase II elongation and chromatin modification complexes. Besides the two core polymerase subunits, members of the PAF complex, SUPT5H, SUPT6H, and FACT complex associated with AID. We show that AID associates with RNA polymerase-associated factor 1 (PAF1) through its N-terminal domain, that depletion of PAF complex members inhibits AID-induced immune diversification, and that the PAF complex can serve as a binding platform for AID on chromatin. A model is emerging of how RNA polymerase II elongation and pausing induce and resolve AID lesions.
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PMID:A role for the RNA pol II-associated PAF complex in AID-induced immune diversification. 2300 33

RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs) in nascent RNA. Our results show that RDDs begin to occur in RNA chains ~55 nt from the RNA polymerase II (Pol II) active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA-editing mechanisms. Moreover, the 55 nt delay in appearance indicates that they do not arise during RNA synthesis by Pol II or as a direct consequence of modified base incorporation. Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in cotranscriptional RNA processing.
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PMID:RNA-DNA differences are generated in human cells within seconds after RNA exits polymerase II. 2456 Dec 52