scholarly journals The DDE Motif in RAG-1 Is Contributed intrans to a Single Active Site That Catalyzes the Nicking and Transesterification Steps of V(D)J Recombination

2001 ◽  
Vol 21 (2) ◽  
pp. 449-458 ◽  
Author(s):  
Patrick C. Swanson
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Active Sites ◽  
Signal Sequence ◽  
Catalytic Triad ◽  
Dna Breaks ◽  
Site Mutation ◽  
Recombination Signal ◽  
Rag 1

ABSTRACT The process of assembling immunoglobulin and T-cell receptor genes from variable (V), diversity (D), and joining (J) gene segments, called V(D)J recombination, involves the introduction of DNA breaks at recombination signals. DNA cleavage is catalyzed by RAG-1 and RAG-2 in two chemical steps: first-strand nicking, followed by hairpin formation via direct transesterification. In vitro, these reactions minimally proceed in discrete protein-DNA complexes containing dimeric RAG-1 and one or two RAG-2 monomers bound to a single recombination signal sequence. Recently, a DDE triad of carboxylate residues essential for catalysis was identified in RAG-1. This catalytic triad resembles the DDE motif often associated with transposase and retroviral integrase active sites. To investigate which RAG-1 subunit contributes the residues of the DDE triad to the recombinase active site, cleavage of intact or prenicked DNA substrates was analyzed in situ in complexes containing RAG-2 and a RAG-1 heterodimer that carried an active-site mutation targeted to the same or opposite RAG-1 subunit mutated to be incompetent for DNA binding. The results show that the DDE triad is contributed to a single recombinase active site, which catalyzes the nicking and transesterification steps of V(D)J recombination by a single RAG-1 subunit opposite the one bound to the nonamer of the recombination signal undergoing cleavage (cleavage intrans). The implications of a trans cleavage mode observed in these complexes on the organization of the V(D)J synaptic complex are discussed.

scholarly journals A RAG-1/RAG-2 Tetramer Supports 12/23-Regulated Synapsis, Cleavage, and Transposition of V(D)J Recombination Signals

2002 ◽  
Vol 22 (22) ◽  
pp. 7790-7801 ◽  
Author(s):  
Patrick C. Swanson
Keyword(s):  
Signal Sequence ◽  
Hmg Proteins ◽  
Mobility Shift ◽  
Recombination Signal ◽  
Recombination Signal Sequences ◽  
Target Dna ◽  
Mobility Shift Assay ◽  
Rag 1 ◽  
Dna Complex

ABSTRACT Initiation of V(D)J recombination involves the synapsis and cleavage of a 12/23 pair of recombination signal sequences by RAG-1 and RAG-2. Ubiquitous nonspecific DNA-bending factors of the HMG box family, such as HMG-1, are known to assist in these processes. After cleavage, the RAG proteins remain bound to the cut signal ends and, at least in vitro, support the integration of these ends into unrelated target DNA via a transposition-like mechanism. To investigate whether the protein complex supporting synapsis, cleavage, and transposition of V(D)J recombination signals utilized the same complement of RAG and HMG proteins, I compared the RAG protein stoichiometries and activities of discrete protein-DNA complexes assembled on intact, prenicked, or precleaved recombination signal sequence (RSS) substrates in the absence and presence of HMG-1. In the absence of HMG-1, I found that two discrete RAG-1/RAG-2 complexes are detected by mobility shift assay on all RSS substrates tested. Both contain dimeric RAG-1 and either one or two RAG-2 subunits. The addition of HMG-1 supershifts both complexes without altering the RAG protein stoichiometry. I find that 12/23-regulated recombination signal synapsis and cleavage are only supported in a protein-DNA complex containing HMG-1 and a RAG-1/RAG-2 tetramer. Interestingly, the RAG-1/RAG-2 tetramer also supports transposition, but HMG-1 is dispensable for its activity.

scholarly journals RAG-1 and RAG-2-dependent assembly of functional complexes with V(D)J recombination substrates in solution.

1997 ◽  
Vol 17 (12) ◽  
pp. 6932-6939 ◽  
Author(s):  
W Li ◽  
P Swanson ◽  
S Desiderio
Keyword(s):  
Dna Cleavage ◽  
Receptor Gene ◽  
Stable Complex ◽  
Dna Breaks ◽  
Cleavage Reaction ◽  
Substrate Complex ◽  
Rag 1 ◽  
The Individual ◽  
Hairpin Formation

V(D)J recombination is initiated by RAG-1 and RAG-2, which introduce double-strand DNA breaks at recombination signal sequences (RSSs) of antigen receptor gene segments to produce signal ends, terminating in blunt, double-strand breaks, and coding ends, terminating in DNA hairpins. While the formation of RAG-RSS complexes has been documented, observations regarding the individual contributions of RAG-1 and RAG-2 to RSS recognition are in conflict. Here we describe an assay for formation and maintenance of functional RAG-RSS complexes in the course of the DNA cleavage reaction. Under conditions of in vitro cleavage, the RAG proteins sequester intact substrate DNA in a stable complex which is formed prior to strand scission. The cleavage reaction subsequently proceeds through nicking and hairpin formation without dissociation of substrate. Notably, the presence of both RAG-1 and RAG-2 is essential for formation of stable, functional complexes with substrate DNA under conditions of the sequestration assay. Two classes of substrate mutation are distinguished by their effects on RAG-mediated DNA cleavage in vitro. A mutation of the first class, residing within the RSS nonamer and associated with coordinate impairment of nicking and hairpin formation, greatly reduces the stability of RAG association with intact substrate DNA. In contrast, a mutation of the second class, lying within the RSS heptamer and associated with selective abolition of hairpin formation, has little or no effect on the half-life of the RAG-substrate complex.

Synthesis, in vitro and in silico studies of inhibitory activity towards α-amylase of bis-azole scaffolds linked by an alkylsulfanyl chain

2020 ◽  
Vol 98 (11) ◽  
pp. 725-735
Author(s):  
Vnira R. Akhmetova ◽  
Nail S. Akhmadiev ◽  
Radik A. Zainullin ◽  
Veronika R. Khayrullina ◽  
Ekaterina S. Mescheryakova ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Water Soluble ◽  
Human Pancreas ◽  
Diethyl Sulfide ◽  
High Affinity ◽  
Competitive Mechanism ◽  
In Silico Studies ◽  
Noncompetitive Inhibitor

Twelve new α,ω-bis[(3,5-dimethylpyrazol-4-yl)methylsulfanyl]alkanes linked by alkyl, diethyl sulfide, and triethyl dioxide spacers were prepared by the multicomponent reaction of acetylacetone, formaldehyde, α,ω-dithiols, and monosubstituted hydrazines. Testing of these products for inhibition of α-amylase enzyme in vitro showed that bis(N-methylpyrazolylmethylsulfanyl)ethane 4a inhibits the enzyme by the competitive mechanism. Meanwhile, the water-soluble adduct of bis(isoxazolylmethylsulfanyl)ethane 2 with HCl (2·HCl) is a noncompetitive inhibitor. The molecular docking results attest to high complementarity between the test molecules and the enzymes such as α-amylases from Aspergillus niger and human pancreas. Bis-pyrazole compounds 1, 1·HCl, and 4a and bis-isoxazole compounds 2 and 2·HCl positioned in the active site of both α-amylases form two closely spaced clusters. For all cases, the bioactive conformations of the modeled ligands were identified, demonstrating high affinity of the bis-azoles (1, 1·HCl, 2, 2·HCl, 4a) to the enzymes. Hydrogen bonds stabilizing their position in both α-amylases active sites were identified.

scholarly journals Sequence of the Spacer in the Recombination Signal Sequence Affects V(D)J Rearrangement Frequency and Correlates with Nonrandom Vκ Usage In Vivo

1998 ◽  
Vol 187 (9) ◽  
pp. 1495-1503 ◽  
Author(s):  
Bertrand Nadel ◽  
Alan Tang ◽  
Guia Escuro ◽  
Geanncarlo Lugo ◽  
Ann J. Feeney
Keyword(s):  
Relative Frequency ◽  
Signal Sequence ◽  
Consensus Sequence ◽  
Recombination Signal Sequence ◽  
Recombination Rates ◽  
Recombination Signal ◽  
Functional Variable ◽  
Determinant Factor

Functional variable (V), diversity (D), and joining (J) gene segments contribute unequally to the primary repertoire. One factor contributing to this nonrandom usage is the relative frequency with which the different gene segments rearrange. Variation from the consensus sequence in the heptamer and nonamer of the recombination signal sequence (RSS) is therefore considered a major factor affecting the relative representation of gene segments in the primary repertoire. In this study, we show that the sequence of the spacer is also a determinant factor contributing to the frequency of rearrangement. Moreover, the effect of the spacer on recombination rates of various human Vκ gene segments in vitro correlates with their frequency of rearrangement in vivo in pre-B cells and with their representation in the peripheral repertoire.

scholarly journals Structural insights into lipoprotein N-acylation byEscherichia coliapolipoprotein N-acyltransferase

2017 ◽  
Vol 114 (30) ◽  
pp. E6044-E6053 ◽  
Author(s):  
Cameron L. Noland ◽  
Michele D. Kattke ◽  
Jingyu Diao ◽  
Susan L. Gloor ◽  
Homer Pantua ◽  
...  
Keyword(s):  
Active Site ◽  
Signal Sequence ◽  
Acyl Chain ◽  
Transmembrane Helix ◽  
Chloride Ion ◽  
Catalytic Triad ◽  
Signal Peptidase ◽  
Membrane Protein Folding ◽  
E Coli ◽  
Structural Insights

Gram-negative bacteria express a diverse array of lipoproteins that are essential for various aspects of cell growth and virulence, including nutrient uptake, signal transduction, adhesion, conjugation, sporulation, and outer membrane protein folding. Lipoprotein maturation requires the sequential activity of three enzymes that are embedded in the cytoplasmic membrane. First, phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) recognizes a conserved lipobox motif within the prolipoprotein signal sequence and catalyzes the addition of diacylglycerol to an invariant cysteine. The signal sequence is then cleaved by signal peptidase II (LspA) to give an N-terminal S-diacylglyceryl cysteine. Finally, apolipoproteinN-acyltransferase (Lnt) catalyzes the transfer of thesn-1-acyl chain of phosphatidylethanolamine to this N-terminal cysteine, generating a mature, triacylated lipoprotein. Although structural studies of Lgt and LspA have yielded significant mechanistic insights into this essential biosynthetic pathway, the structure of Lnt has remained elusive. Here, we present crystal structures of wild-type and an active-site mutant ofEscherichia coliLnt. The structures reveal a monomeric eight-transmembrane helix fold that supports a periplasmic carbon–nitrogen hydrolase domain containing a Cys–Glu–Lys catalytic triad. Two lipids are bound at the active site in the structures, and we propose a putative phosphate recognition site where a chloride ion is coordinated near the active site. Based on these structures and complementary cell-based, biochemical, and molecular dynamics approaches, we propose a mechanism for substrate engagement and catalysis byE. coliLnt.

scholarly journals RAG Transposase Can Capture and Commit to Target DNA before or after Donor Cleavage

2001 ◽  
Vol 21 (13) ◽  
pp. 4302-4310 ◽  
Author(s):  
Matthew B. Neiditch ◽  
Gregory S. Lee ◽  
Mark A. Landree ◽  
David B. Roth
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Cell Receptor ◽  
Rate Limiting Step ◽  
Target Capture ◽  
Target Dna ◽  
Before And After ◽  
Rag 1 ◽  
Antigen Receptor Loci

ABSTRACT The discovery that the V(D)J recombinase functions as a transposase in vitro suggests that transposition by this system might be a potent source of genomic instability. To gain insight into the mechanisms that regulate transposition, we investigated a phenomenon termed target commitment that reflects a functional association between the RAG transposase and the target DNA. We found that the V(D)J recombinase is quite promiscuous, forming productive complexes with target DNA both before and after donor cleavage, and our data indicate that the rate-limiting step for transposition occurs after target capture. Formation of stable target capture complexes depends upon the presence of active-site metal binding residues (the DDE motif), suggesting that active-site amino acids in RAG-1 are critical for target capture. The ability of the RAG transposase to commit to target prior to cleavage may result in a preference for transposition into nearby targets, such as immunoglobulin and T-cell receptor loci. This could bias transposition toward relatively “safe” regions of the genome. A preference for localized transposition may also have influenced the evolution of the antigen receptor loci.

scholarly journals Active-Site Models of Streptococcus pyogenes Cas9 in DNA Cleavage State

2021 ◽  
Vol 8 ◽  
Author(s):  
Honghai Tang ◽  
Hui Yuan ◽  
Wenhao Du ◽  
Gan Li ◽  
Dongmei Xue ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Active Sites ◽  
Structural Similarity ◽  
Atomic Model ◽  
Nucleophilic Attack ◽  
Target Dna ◽  
Coordinated Water ◽  
Dna Complex

CRISPR-Cas9 is a powerful tool for target genome editing in living cells. Significant advances have been made to understand how this system cleaves target DNA. HNH is a nuclease domain, which shares structural similarity with the HNH endonuclease characterzied by a beta-beta-alpha-metal fold. Therefore, based on one- and two-metal-ion mechanisms, homology modeling and molecular dynamics (MD) simulation are suitable tools for building an atomic model of Cas9 in the DNA cleavage state. Here, by modeling and MD, we presented an atomic model of SpCas9–sgRNA–DNA complex with the cleavage state. This model shows that the HNH and RuvC conformations resemble their DNA cleavage state where the active-sites in the complex coordinate with DNA, Mg2+ ions, and water. Among them, residues D10, E762, H983, and D986 locate at the first shell of the RuvC active-site and interact with the ions directly, residues H982 or/and H985 are general (Lewis) bases, and the coordinated water is located at the positions for nucleophilic attack of the scissile phosphate. Meanwhile, this catalytic model led us to engineer a new SpCas9 variant (SpCas9-H982A + H983D) with reduced off-target effects. Thus, our study provided new mechanistic insights into the CRISPR-Cas9 system in the DNA cleavage state and offered useful guidance for engineering new CRISPR-Cas9 editing systems with improved specificity.

The recombination signal sequence-binding protein RBP-2N functions as a transcriptional repressor

1994 ◽  
Vol 14 (5) ◽  
pp. 3310-3319
Author(s):  
S Dou ◽  
X Zeng ◽  
P Cortes ◽  
H Erdjument-Bromage ◽  
P Tempst ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Signal Sequence ◽  
Dna Recognition ◽  
Cellular Protein ◽  
Recombination Signal Sequence ◽  
Recognition Sequence ◽  
General Role ◽  
Recombination Signal

We have identified a cellular protein, RBP-2N, a presumed recombinase, as a repressor of transcription. Inhibition of transcription by RBP-2N was dependent on its DNA recognition site and was demonstrated in vitro and in vivo. This repression appears to be general, as transcription mediated by SP1 and Gal4/VP16 was inhibited by RBP-2N. The protein was purified to near homogeneity from human cells on the basis of its binding to a site present in the promoter of the adenovirus pIX gene. The DNA recognition sequence is 5'-TGGGAAAGAA, which is markedly different from the recombination signal sequence originally identified as the target site for this protein. The sequence of the purified protein is 97% identical with that published for the mouse RBP-2N protein. The reported homolog in Drosophila is Suppressor of Hairless. RBP-2N binding sites are present in a number of cellular and viral promoters, so RBP-2N may have a general role in transcriptional repression.

scholarly journals Molecular mechanism for generation of antibody memory

2008 ◽  
Vol 364 (1517) ◽  
pp. 569-575 ◽  
Author(s):  
Velizar Shivarov ◽  
Reiko Shinkura ◽  
Tomomitsu Doi ◽  
Nasim A Begum ◽  
Hitoshi Nagaoka ◽  
...  
Keyword(s):  
Rna Editing ◽  
Dna Cleavage ◽  
De Novo ◽  
Alternative Hypothesis ◽  
Basic Site ◽  
Immunoglobulin Gene ◽  
Dna Breaks ◽  
Dna Deamination

Activation-induced cytidine deaminase (AID) is the essential enzyme inducing the DNA cleavage required for both somatic hypermutation and class switch recombination (CSR) of the immunoglobulin gene. We originally proposed the RNA-editing model for the mechanism of DNA cleavage by AID. We obtained evidence that fulfils three requirements for CSR by this model, namely (i) AID shuttling between nucleus and cytoplasm, (ii) de novo protein synthesis for CSR, and (iii) AID–RNA complex formation. The alternative hypothesis, designated as the DNA-deamination model, assumes that the in vitro DNA deamination activity of AID is representative of its physiological function in vivo . Furthermore, the resulting dU was removed by uracil DNA glycosylase (UNG) to generate a basic site, followed by phosphodiester bond cleavage by AP endonuclease. We critically examined each of these provisional steps. We identified a cluster of mutants (H48A, L49A, R50A and N51A) that had particularly higher CSR activities than expected from their DNA deamination activities. The most striking was the N51A mutant that had no ability to deaminate DNA in vitro but retained approximately 50 per cent of the wild-type level of CSR activity. We also provide further evidence that UNG plays a non-canonical role in CSR, namely in the repair step of the DNA breaks. Taking these results together, we favour the RNA-editing model for the function of AID in CSR.

scholarly journals Functional and computational identification of a rescue mutation near the active site of an mRNA methyltransferase

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Pierre-Yves Colin ◽  
Paul A. Dalby
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Large Scale ◽  
Interaction Network ◽  
Catalytic Efficiency ◽  
Transferase Activity ◽  
Viral Mrna ◽  
In Vitro Production ◽  
Cell Immunity

AbstractRNA-based drugs are an emerging class of therapeutics combining the immense potential of DNA gene-therapy with the absence of genome integration-associated risks. While the synthesis of such molecules is feasible, large scale in vitro production of humanised mRNA remains a biochemical and economical challenge. Human mRNAs possess two post-transcriptional modifications at their 5′ end: an inverted methylated guanosine and a unique 2′O-methylation on the ribose of the penultimate nucleotide. One strategy to precisely methylate the 2′ oxygen is to use viral mRNA methyltransferases that have evolved to escape the host’s cell immunity response following virus infection. However, these enzymes are ill-adapted to industrial processes and suffer from low turnovers. We have investigated the effects of homologous and orthologous active-site mutations on both stability and transferase activity, and identified new functional motifs in the interaction network surrounding the catalytic lysine. Our findings suggest that despite their low catalytic efficiency, the active-sites of viral mRNA methyltransferases have low mutational plasticity, while mutations in a defined third shell around the active site have strong effects on folding, stability and activity in the variant enzymes, mostly via network-mediated effects.

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