Crystal structure of the V(D)J recombinase RAG1–RAG2

Kim, Min Sung; Lapkouski, Mikalai; Yang, Wei; Gellert, Martin

doi:10.1038/nature14174

Cited by 148 publications

(212 citation statements)

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“…The relative accessibility of RAG-1 cysteine residues to pulse alkylation in a native protein preparation incubated with the H3K4me0 control showed good correlation with the computed solvent-accessible area of cysteine residues in two available RAG structures (11,20) (Fig. S3 and Table S1).…”

Section: Resultsmentioning

confidence: 78%

“…S3 and Table S1). The accessibility of RAG-1 C431 to pulse alkylation was more closely correlated with its solvent-accessible area in the electron microscopic structure (20) than in the crystallographic structure (11), suggesting that the former structure may more closely approximate the predominant solution conformation of the NBD in the absence of H3K4me3 (Fig. S3 A-D).…”

Section: Resultsmentioning

confidence: 95%

mentioning

confidence: 99%

“…The catalytic core and DNA-binding functions are largely contained within RAG-1 (11). RAG-2, which is also essential for DNA cleavage activity, comprises part of a putative DNA-binding cleft and participates in formation of the active site (11). Although residues 387 through 527 of RAG-2 are dispensable for DNA cleavage in vitro, this region supports several regulatory functions in vivo, including binding of RAG-2 to H3K4me3 (7,8,12).…”

mentioning

confidence: 99%

“…mass spectrometry (18). Strikingly, binding of H3K4me3 to RAG-2 is accompanied by robust increases in the solvent accessibility of RAG-1 within the dimerization and DNA-binding domain (DDBD) and in the ZnH2 domain, which acts as a scaffold for the catalytic center (11). These H3K4me3-induced changes in solvent accessibility are abolished by mutation of the RAG-2 PHD finger.…”

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confidence: 99%

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H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase

Bettridge

Pandey

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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V(D)J recombination is initiated by the recombination-activating gene (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 stimulates substrate binding and catalysis, which are functions of RAG-1. This has suggested an allosteric mechanism in which information regarding occupancy of the RAG-2 PHD is transmitted to RAG-1. To determine whether the conformational distribution of RAG is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols by differential isotopic chemical footprinting. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within a DNA-binding domain and in the ZnH2 domain, which acts as a scaffold for the catalytic center. Thus, engagement of H3K4me3 by the RAG-2 PHD is associated with dynamic conformational changes in RAG-1, consistent with allosteric control by active chromatin.DNA recombination | genomic plasticity | allosteric control | epigenetic modification | immune development A ll forms of DNA processing-replication, transcription, recombination, and repair-use allosteric regulation, often as a basis for molecular discrimination but also to establish a sequence of interactions or to bias the outcome of a reaction. In many instances the allosteric ligand is a specific DNA structure, as in Cre-mediated recombination, in which the Holliday junction intermediate effects allosteric conformational changes that switch active and inactive Cre monomers (1). In other instances the allosteric ligand is a DNA-bound protein array, as in λ integration, which is driven to completion by a flanking DNA-protein array that biases the conformation of λ-integrase (2).V(D)J recombination, the process by which antigen receptor genes are assembled, is also subject to allosteric control, but in this case the allosteric ligand is a specific chromatin mark rather than a DNA structure. V(D)J recombination is initiated by recombinationactivating gene (RAG)-1 and RAG-2, which together cleave DNA at recombination signal sequences (RSSs) flanking the participating gene segments (3). There are two classes of RSS, termed 12-RSS and 23-RSS, in which heptamer and nonamer elements are separated by spacers of 12 bp or 23 bp; physiological DNA cleavage requires the pairing of a 12-RSS with a 23-RSS (3). V(D)J recombination acts in an ordered, locus-specific fashion during lymphoid development. The accessibility of gene segments to V(D)J recombination is positively correlated with transcription at the unrearranged locus and with histone modifications characteristic of active chromatin, including hypermethylation of histone H3 at lysine 4 (H3K4me3) (4-10).RAG-1 and RAG-2 are 1,040 and 527 aa residues long, respectively. The catalytic core and DNA-binding functions are largely contained within RAG-1 (11). RAG-2, which is also ess...

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Section: Resultsmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 95%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase

Bettridge

Pandey

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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B‐Cell Antigen Receptor: Assembly and Diversification

Mohammad¹,

Guillou²,

Ghamlouch³

et al. 2020

Encyclopedia of Life Sciences

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Immunoglobulin (Ig) genes of mammalian B cells undergo two processes that diversify their genomic sequence and structure. The first one concerns the BCR assembly that is achieved through a combinatorial rearrangement of a large number of V, D and J gene segments. This process is essential for B‐cell development and occurs during early B‐cell differentiation in the bone marrow without antigenic challenge. The second process includes somatic hypermutation (SHM), and class switch recombination (CSR), start when the B cell encounters an antigen at the periphery. While the two processes share the action of ubiquitous DNA repair factors to carry out all the steps necessary for creating recombination and mutations in genomic DNA, they are initiated by different lymphoid‐specific factors. On one hand, the action of recombination activating genes 1 and 2 (RAG1 and RAG2) is required for the V(D) J rearrangements and, on the other hand, the action of activation‐induced cytidine deaminase (AID) for both SHM and CSR. The ultimate goal of these diversification mechanisms is to create a dynamic and robust immune response. Key Concepts V(D)J recombination is a programmed that generates B‐ and T‐cell receptors in vertebrates (BCR and TCR). V(D)J recombination requires precise cutting of the DNA at recombination signal sequences (RSS) followed by rejoining of the resulting termini. Imprecisions during the ends‐joining reaction contribute significantly to increasing the variability of the resulting functional BCR and TCR. RAG1, RAG2 and TdT are the three lymphoid‐specific factors needed for V(D)J recombination and for the junctional diversity observed during lymphoid development. B‐cells provide the antibody‐mediated immune response (humoral immunity). It can generate antibodies to an immense variety of pathogenic antigens. Somatic hypermutation (SHM) and class switch recombination (CSR) occur only in germinal centre B cells. During SHM, DNA repair mechanisms are diverted from their canonical role in preserving genomic integrity to permit high rate of mutations. SHM and CSR both occur in germinal centre in secondary lymphoid tissues and require activation‐induced cytidine deaminase (AID). AID deaminates deoxy‐cytosine on single‐stranded DNA. Ten–eleven translocation (TET) proteins (mainly TET2et TET3) are key regulators of immunoglobulin light chain rearrangement, class switch recombination and plasma cell differentiation.

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Novel molecular mechanism for generating NK‐cell fitness and memory

Karo

Sun

2015

Eur J Immunol

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The mammalian immune system has been traditionally subdivided into two compartments known as the innate and the adaptive. T cells and B cells, which rearrange their antigen receptor genes using the RAG recombinase, comprise the adaptive arm of immunity. Meanwhile, every other white blood cell has been grouped together under the broad umbrella of innate immunity, including natural killer (NK) cells. NK cells are considered innate lymphocytes because of their rapid responses to stressed cells and their ability to develop without receptor gene rearrangement (i.e. in RAG-deficient mice). However, new findings implicate a critical function for RAG proteins during NK cell ontogeny, and suggest a novel mechanism by which controlled DNA breaks during NK cell development dictate the fitness, function, and longevity of these cells. This review highlights recent work describing how DNA break events can impact cellular differentiation and fitness in a variety of cell types and settings.

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Crystal structure of the V(D)J recombinase RAG1–RAG2

Cited by 148 publications

References 61 publications

H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase

H3K4me3 induces allosteric conformational changes in the DNA-binding and catalytic regions of the V(D)J recombinase

B‐Cell Antigen Receptor: Assembly and Diversification

Novel molecular mechanism for generating NK‐cell fitness and memory

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