Simultaneous 13C and 15N isotope editing of biomolecular complexes. Application to a mutant lac repressor headpiece DNA complex.
Explore the source record for details and available documents.
Carbon↗
SEARCH · Search PubMed
Results for “DNA editing”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
The solution structure of the Zalpha domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA.
Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Zalpha. Here we report the solution structure of free Zalpha and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Zalpha)(2)/Z-DNA complex shows that most Z-DNA contacting residues in free Zalpha are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (alpha+beta)helix-turn-helix/B-DNA complexes suggests that binding of Zalpha to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B- and Z-DNA.
Adenosine Deaminase↗
A quantitative method to detect RNA editing events.
Explore the source record for details and available documents.
Animals↗
Unexplained complexity of the mitochondrial genome and transcriptome in kinetoplastid flagellates.
Kinetoplastids are flagellated protozoans, whose members include the pathogens Trypanosoma brucei, T. cruzi and Leishmania species, that are considered among the earliest diverging eukaryotes with a mitochondrion. This organelle has become famous because of its many unusual properties, which are unique to the order Kinetoplastida, including an extensive kinetoplast DNA network and U-insertion/deletion type RNA editing of its mitochondrial transcripts. In the last decade, considerable progress has been made in elucidating the complex machinery of RNA editing. Moreover, our understanding of the structure and replication of kinetoplast DNA has also dramatically improved. Much less however, is known, about the developmental regulation of RNA editing, its integration with other RNA maturation processes, stability of mitochondrial mRNAs, or evolution of the editing process itself. Yet the profusion of genomic data recently made available by sequencing consortia, in combination with methods of reverse genetics, hold promise in understanding the complexity of this exciting organelle, knowledge of which may enable us to fight these often medically important protozoans.
Animals↗
Hydrolytic nucleoside and nucleotide deamination, and genetic instability: a possible link between RNA-editing enzymes and cancer?
Post-transcriptional RNA editing generates novel gene products by changing the coding sequence of the transcript from that in the genome. Two classes of RNA editing exist in mammals, each of which involves an enzymatic deamination. These reactions have stringent sequence and structural requirements for their target RNAs, and each requires distinctive enzymatic machinery. Alterations in the expression or abundance of RNA-editing factors produce unanticipated alterations in the processing or expression of RNAs, in some cases outside their physiological targets. Recent findings suggest that unregulated expression of the cytidine-deaminase gene family might lead to deamination of deoxycytidine nucleotides in DNA. Aberrant or dysregulated RNA editing, or altered expression of editing factors, might contribute to genomic instability in cancer.
Animals↗
The bacteriophage phi 29 DNA polymerase, a proofreading enzyme.
The bacteriophage phi 29 DNA polymerase, involved both in the protein-primed initiation and elongation steps of the viral DNA replication, displays a very processive 3',5'-exonuclease activity acting preferentially on single-stranded DNA. This exonucleolytic activity showed a marked preference for excision of a mismatched versus a correctly paired 3' terminus. These characteristics enable the phi 29 DNA polymerase to act as a proofreading enzyme. A comparative analysis of the wild-type phi 29 DNA polymerase and a mutant lacking 3',5'-exonuclease activity indicated that a productive coupling between the exonuclease and polymerase activities is necessary to prevent fixation of polymerization errors. Based on these data, the phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication, appears to share the same mechanism for the editing function as that first proposed for T4 DNA polymerase and Escherichia coli DNA polymerase I on the basis of functional and structural studies.
Bacteriophages↗
High levels of RNA editing in a vascular plant chloroplast genome: analysis of transcripts from the fern Adiantum capillus-veneris.
We sequenced transcripts from all putative genes for proteins, rRNAs, and a selection of gene-encoding tRNAs in the chloroplast genome of the fern Adiantum capillus-veneris. We detected 350 RNA editing sites when the cDNA sequence was compared to that of the genomic DNA. Of these sites, 10% were U-to-C edits and 90% were C-to-U edits. RNA editing created 19 new start codons, three new stop codons, and "repaired" 26 internal stop codons. Of the 332 editing sites that altered a codon, 26% were in the first codon position, 68% in the second, and 6% in the third. We also detected 21 silent edits, as well as 19 edits that were in untranslated regions, including introns and the anticodon of tRNA(Leu). The latter edit provided a tRNA that is not otherwise encoded in this genome and accounts for a heavily used leucine codon. The level of RNA editing in this fern is more than ten times that of any other vascular plant examined across an entire chloroplast genome. A previous study found even higher levels of editing in a hornwort (942 sites). This suggests that the relatively low levels of editing in seed plants (less than 0.05%) may not be typical for land plants, and that RNA editing may play a major role in chloroplast genome processing. Additionally, we found that 53 editing sites in A. capillus-veneris are homologous to editing sites in the hornwort, and some other land plants. This implies that a major component of RNA editing sites have been conserved for hundreds of millions of years.
Adiantum↗
Sequence data handling by computer.
The speed of the new DNA sequencing techniques has created a need for computer programs to handle the data produced. This paper describes simple programs designed specifically for use by people with little or no computer experience. The programs are for use on small computers and provide facilities for storage, editing and analysis of both DNA and amino acid sequences. A magnetic tape containing these programs is available on request.
Amino Acid Sequence↗
Characterisation of kinetoplast DNA minicircles from Herpetomonas samuelpessoai.
In this study, we have sequenced more than 100 clones of minicircle DNA from Herpetomonas samuelpessoai. An unusual amplification approach was developed to amplify minicircle DNA by using a pair of complementary primers designed from a universal stretch of minicircle sequence. Sequence analysis shows that the kinetoplast minicircles in Herpetomonas with a size of 1.3 kb are organised into two conserved regions and two variable regions which are located 180 degrees apart. The potential gRNA genes are encoded in variable regions of minicircle approximately 360 bp from CSB-3 (conserved sequence block 3). A conserved upstream sequence located 30 nt before the gRNA genes was identified and is related to the gRNA genes in sequence organisation. A potential role(s) of this sequence in gRNA transcription is discussed.
Animals↗
The application of numerical estimates of base calling accuracy to DNA sequencing projects.
During DNA sequencing projects one of the most labour intensive and highly skilled tasks is to view the original trace descriptions of gels and to adjudicate between conflicting readings. Given the current methods of calculating a consensus, the majority of the time employed in viewing traces and editing readings is actually devoted to making the poorer data fit the good data. We propose new consensus calculation algorithms that employ numerical estimates of base calling accuracy and which when used in conjunction with an automatic detector of contradictory data should greatly reduce the time spent checking and editing readings and hence improve DNA sequencing productivity.
Algorithms↗
Involvement of 5' flanking sequence for specifying RNA editing sites in plant mitochondria.
Unsuccessful insertion of foreign DNA into plant mitochondrial genomes has hindered scientific evaluation of cis-elements needed for RNA editing. Both a normal atp6 gene and a chimeric atp6 sequence are present in rice mitochondria. The chimeric atp6 contains one-half of the normal atp6 sequence in its 5' portion and an unknown sequence in its downstream portion. The C-nucleotide at position 511, located just upstream of the unknown sequence recombined in the chimeric atp6 sequence, is edited, as are other possible editing sites upstream from position 511. We report here that the 5' sequence adjacent to the editing site of atp6 contains cis-information required for RNA editing and that the 3' sequence flanking the editing site provides little contribution to editing-site recognition.
Amino Acid Sequence↗
Editing of pre-mRNAs can occur before cis- and trans-splicing in Petunia mitochondria.
Plant mitochondrial mRNAs have recently been shown to undergo editing, involving cytidine-to-uridine changes relative to the DNA sequence. We have examined the temporal relationship of editing and intron removal in coxII mRNAs in Petunia mitochondria. By using differential hybridization to probes specific for edited and unedited RNA and by sequencing of individual unspliced coxII pre-mRNA cDNAs, we found that RNA editing at any editing site can precede the splicing event. Similar results were obtained from examinations of pre-mRNA cDNAs of nad1, a gene composed of multiple exons that are both cis and trans spliced. Thus, intron removal is not required before editing can occur. The existence of editing intermediates indicates that the editing process is not strictly coincident with transcription.
Antisense Elements (Genetics)↗
Generation and analysis of 25 Mb of genomic DNA from the pufferfish Fugu rubripes by sequence scanning.
We have generated and analyzed >50,000 shotgun clones from 1059 Fugu cosmid clones. All sequences have been minimally edited and searched against protein and DNA databases. These data are all displayed on a searchable, publicly available web site at. With an average of 50 reads per cosmid, this is virtually nonredundant sequence skimming, covering 30%-50% of each clone. This essentially random data set covers nearly 25 Mb (>6%) of the Fugu genome and forms the basis of a series of whole genome analyses which address questions regarding gene density and distribution in the Fugu genome and the similarity between Fugu and mammalian genes. The Fugu genome, with eight times less DNA but a similar gene repertoire, is ideally suited to this type of study because most cosmids contain more than one identifiable gene. General features of the genome are also discussed. We have made some estimation of the syntenic relationship between mammals and Fugu and looked at the efficacy of ORF prediction from short, unedited Fugu genomic sequences. Comparative DNA sequence analyses are an essential tool in the functional interpretation of complex vertebrate genomes. This project highlights the utility of using the Fugu genome in this kind of study.
Animals↗
B cells from hyper-IgM patients carrying UNG mutations lack ability to remove uracil from ssDNA and have elevated genomic uracil.
The generation of high-affinity antibodies requires somatic hypermutation (SHM) and class switch recombination (CSR) at the immunoglobulin (Ig) locus. Both processes are triggered by activation-induced cytidine deaminase (AID) and require UNG-encoded uracil-DNA glycosylase. AID has been suggested to function as an mRNA editing deaminase or as a single-strand DNA deaminase. In the latter model, SHM may result from replicative incorporation of dAMP opposite U or from error-prone repair of U, whereas CSR may be triggered by strand breaks at abasic sites. Here, we demonstrate that extracts of UNG-proficient human B cell lines efficiently remove U from single-stranded DNA. In B cell lines from hyper-IgM patients carrying UNG mutations, the single-strand-specific uracil-DNA glycosylase, SMUG1, cannot complement this function. Moreover, the UNG mutations lead to increased accumulation of genomic uracil. One mutation results in an F251S substitution in the UNG catalytic domain. Although this UNG form was fully active and stable when expressed in Escherichia coli, it was mistargeted to mitochondria and degraded in mammalian cells. Our results may explain why SMUG1 cannot compensate the UNG2 deficiency in human B cells, and are fully consistent with the DNA deamination model that requires active nuclear UNG2. Based on our findings and recent information in the literature, we present an integrated model for the initiating steps in CSR.
B-Lymphocytes↗
SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.
Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.
Humans↗
Base editing for precision therapeutics.
Base editing (BE), the precise installation of single-nucleotide changes in DNA or RNA without inducing double-strand breaks, holds substantial therapeutic promise for correcting single-nucleotide variants, which constitute more than half of the known pathogenic genetic variants. Recent advances have improved base editor specificity, efficiency, and delivery, enabling clinically oriented procedures. Clinically, BE has shown early success or strong translational promise in sickle cell disease, β-thalassemia, leukemia (via CAR T and epitope engineering), hypercholesterolemia (PCSK9 and ANGPTL3), alpha-1-antitrypsin deficiency, and glycogen storage disease type Ia. Key remaining challenges include bystander editing within the activity window, residual off-target DNA and RNA editing, delivery constraints (payload size, tissue targeting, and redosing limits), immunogenicity, and the need for durable long-term safety evidence across relevant cell types and disease contexts. Continued technological refinements, careful preclinical validation, and rigorous clinical assessment will be essential to fully realize BE's transformative potential in precision medicine.
Humans↗
15N-edited three-dimensional NOESY-HMQC with water flipback: enhancement of weak labile 1H resonances of protein side chains contacting DNA.
Two pulse sequences are described that employ a modified water flipback technique to enhance the signal intensity of weak side chain resonances at the protein-DNA interface of the vnd/NK-2 homeodomain/DNA complex in an 15N-edited three-dimensional NOESY-HMQC spectrum. The pulse sequences presented employ water flipback pulses at the beginning of the NOESY mixing time, optimizing the direct NOE transfer of magnetization from the water to the protein by maximizing the z-component of the water magnetization. In one of the pulse sequences, radiation damping during the the indirect 1H and 15N evolution times is suppressed. A modified version of the WATERGATE water suppression technique is employed during the HMQC portion of the experiment. The signal enhancement is demonstrated for the resonances of the side chain amide of Asn51, an invariant homeodomain residue whose contact with the DNA is critical for binding. An ancillary advantage of the experiment is the ability to observe NOE transfer of magnetization from water. The information present in the water resonance plane of the three-dimensional spectrum is illustrated in a comparison with the corresponding HMQC spectrum of the protein/DNA complex.
Binding Sites↗
5-HT(2C) receptor RNA editing in the amygdala of C57BL/6J, DBA/2J, and BALB/cJ mice.
Post-transcriptional RNA editing of the G-protein coupled 5-hydroxytryptamine-2C (5-HT(2C)) receptor predicts an array of 24 receptor isoforms, some of which are characterized by reduced constitutive activity and potency to initiate intracellular signaling. The amygdala is integral to anxiety, fear, and related psychiatric diseases. Activation of 5-HT(2C) receptors within the amygdala is anxiogenic. Here, we describe the RNA editing profiles from amygdala of two inbred mouse strains (BALB/cJ and DBA/2J) known to be more anxious than a third (C57BL/6J). We confirmed the strain anxiety differences using light<-->dark exploration, and we discovered that BALB/cJ and DBA/2J are each characterized by a higher functioning RNA editing profile than C57BL/6J. BALB/cJ and DBA/2J exhibit a roughly two-fold reduction in C site editing, and a corresponding two-fold reduction in the edited isoform VSV. C57BL/6J is characterized by a relative decrease in the unedited highly functional isoform INI. We estimated the heritability of editing at the C site to be approximately 40%. By sequencing genomic DNA, we found complete conservation between C57BL/6J, BALB/cJ, DBA/2J and 37 other inbred strains for the RNA edited region of Htr2c, suggesting Htr2c DNA sequence does not influence variation in Htr2c RNA editing between inbred strains of mice. We did, however, discover that serotonin turnover is reduced in BALB/cJ and DBA/2J, consistent with emerging evidence that synaptic serotonin levels regulate RNA editing. These results encourage further study of the causes and consequences of 5-HT(2C) receptor RNA editing in the amygdala of mice.
Amygdala↗