Cracking the DNA Code for V(D)J Recombination

Kim, M.S; Chuenchor, W.; Chen, X; Cui, Yanxiang; Zhang, X; Zhou, Z. Hong; Gellert, Martin; Yang, Wei

doi:10.1016/j.molcel.2018.03.008

Cited by 65 publications

(134 citation statements)

References 62 publications

(82 reference statements)

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“…Of more than 400 DNA tethers with the 12RSS containing a T-to-A change at heptamer position 6, we observed the PC only once, which subsequently led to cleavage. This result is consistent with recent findings that the consensus TG dinucleotide at the last two positions of the heptamer supports a kink in the DNA and may be critical for RAG binding (14). We notice that some changes increase the median time spent in the PC such as with the heptamer position 4 ( Fig.…”

Section: Resultssupporting

confidence: 93%

“…We also studied changes made to positions 3-7 of the heptamer and various positions of the nonamer. The first three positions of the heptamer are perfectly conserved (7), likely to support DNA distortions needed for nicking and for base-specific interactions with the cleavage domain on RAG1 after nicking (13)(14)(15), while heptamer positions 4-7 also mediate base-specific interactions with RAG (13). The nonamer is bound by a nonamer-specific binding domain on and are drawn before examining the bead trajectories and based on the length of the DNA tether and the distance between the RSSs along the strand, the extent to which HMGB1, a protein that binds nonspecifically to DNA and helps facilitate RAG binding, kinks the DNA and a set of calibration experiments relating the range of motion of the bead to the length of its tether.…”

Section: Resultsmentioning

confidence: 99%

“…rate of completing recombination (8)(9)(10)(11)(12). Recent structural results provide evidence that RAG binding is sensitive to basespecific contacts and the local flexibility or rigidity of the 12and 23RSS (13)(14)(15). Despite this extensive characterization on the interaction, little is known about how a given RSS sequence affects each step of the RAG-RSS reaction.…”

mentioning

confidence: 99%

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Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination

Hirokawa¹,

Chure

Belliveau

et al. 2019

Preprint

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Developing lymphocytes in the immune system of jawed vertebrates assemble antigen-receptor genes by undergoing large-scale reorganization of spatially separated V, D, and J gene segments through a process known as V(D)J recombination. The RAG protein initiates this process by binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12-or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG-RSS interaction. We employ a single-molecule method known as tethered particle motion to quantify the formation, stability, and cleavage of the RAG-12RSS-23RSS paired complex (PC) for numerous synthetic and endogenous 12RSSs. We thoroughly investigate the sequence space around a RSS by making 40 different single-bp changes and characterizing the reaction dynamics. We reveal that single-bp changes affect RAG function based on their position: loss of cleavage function (first three positions of the heptamer); reduced propensity for forming the PC (the nonamer and last four bp of the heptamer); or variable effects on PC formation (spacer). We find that the rare usage of some endogenous gene segments can be mapped directly to their adjacent 12RSSs to which RAG binds weakly. The 12RSS, however, cannot explain the high-frequency usage of other gene segments. Finally, we find that RSS nicking, while not required for PC formation, substantially stabilizes the PC. Our findings provide detailed insights into the contribution of individual RSS positions to steps of the RAG-RSS reaction that previously have been difficult to assess quantitatively. V(D)J recombination | recombination-activating gene | recombination signal sequence | single-molecule biophysics

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination

Hirokawa¹,

Chure

Belliveau

et al. 2019

Preprint

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“…The crystal and cryo‐electron microscopy structures of RAG1/RAG2 core proteins have revealed a Y‐shaped structure, with the RAG1 NBD dimer forming the stem of the Y, while additional regions along the central and C‐terminal region of RAG1 act as DNA‐binding surfaces for the RSS heptamer and coding flank. The three catalytic residues (D603, D711, and E965) reside in the RNH domain and together with the carboxy‐terminal domain form the crevice of the Y structure . The RAG2 core region folds in six‐bladed β‐propeller associates with the RAG1 domains downstream of the DDBD, creating the arms of the Y structure.…”

Section: Structure Of the Human Rag 1 And Rag2 Proteinsmentioning

confidence: 99%

“…The three catalytic residues (D603, D711, and E965) reside in the RNH domain and together with the carboxy-terminal domain form the crevice of the Y structure. 37 The RAG2 core region folds in six-bladed βpropeller associates with the RAG1 domains downstream of the DDBD, creating the arms of the Y structure.…”

Section: Phosphorylation Of Residue T490 By the Cyclin A-cdk2 Complexmentioning

confidence: 99%

RAG gene defects at the verge of immunodeficiency and immune dysregulation

Villa

Notarangelo

2018

Immunological Reviews

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Summary Mutations of the Recombinase Activating Genes (RAG) in humans underlie a broad spectrum of clinical and immunological phenotypes that reflect different degrees of impairment of T and B cell development and alterations of mechanisms of central and peripheral tolerance. Recent studies have shown that this phenotypic heterogeneity correlates, albeit imperfectly, with different levels of recombination activity of the mutant RAG proteins. Furthermore, studies in patients and in newly developed animal models carrying hypomorphic RAG mutations have disclosed various mechanisms underlying immune dysregulation in this condition. Careful annotation of clinical outcome and immune reconstitution in RAG-deficient patients who have received hematopoietic stem cell transplantation has shown that progress has been made in the treatment of this disease, but new approaches remain to be tested to improve stem cell engraftment and durable immune reconstitution. Finally, initial attempts have been made to treat RAG deficiency with gene therapy.

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RAG deficiencies: Recent advances in disease pathogenesis and novel therapeutic approaches

2021

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The RAG1 and RAG2 proteins initiate the process of V(D)J recombination and therefore play an essential role in adaptive immunity. While null mutations in the RAG genes cause severe combined immune deficiency with lack of T and B cells (T − B − SCID) and susceptibility to life-threatening, early-onset infections, studies in humans and mice have demonstrated that hypomorphic RAG mutations are associated with defects of central and peripheral tolerance resulting in immune dysregulation. In this review, we provide an overview of the extended spectrum of RAG deficiencies and their associated clinical and immunological phenotypes in humans. We discuss recent advances in the mechanisms that control RAG expression and function, the effects of perturbed RAG activity on lymphoid development and immune homeostasis, and propose novel approaches to correct this group of disorders.

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Cracking the DNA Code for V(D)J Recombination

Cited by 65 publications

References 62 publications

Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination

Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination

RAG gene defects at the verge of immunodeficiency and immune dysregulation

RAG deficiencies: Recent advances in disease pathogenesis and novel therapeutic approaches

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