The roles of the RAG1 and RAG2 “non-core” regions in V(D)J recombination and lymphocyte development

Jones, Jessica M.; Simkus, Carrie

doi:10.1007/s00005-009-0011-3

Cited by 52 publications

(61 citation statements)

References 85 publications

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“…Rare experiments describing full-length RAG1/2 complexes have reported very low enzyme activities, making conclusions very difficult (12,37). However, recent studies have revealed that noncore regions of RAG proteins have important roles in regulating V(D)J recombination (20). The PHD finger of RAG2 located in the noncore region directly interacts with histone H3, with trimethylation on Lys4 (24,25), and this interaction stimulates RAG cleavage activity, thereby resulting in improved efficiencies of V(D)J recombination (36).…”

Section: Resultsmentioning

confidence: 99%

Mechanistic Basis for RAG Discrimination between Recombination Sites and the Off-Target Sites of Human Lymphomas

Shimazaki

Askary

Swanson

et al. 2012

Molecular and Cellular Biology

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During V(D)J recombination, RAG targeting to correct sites versus off-target sites relies on both DNA sequence features and on chromatin marks. Kinetic analysis using the first highly active full-length purified RAG1/RAG2 complexes has now allowed us to define the important catalytic features of this complex. We found that the overall rate of nicking, but not hairpinning, is critical for the discrimination between correct (optimal) versus off-target (suboptimal) sites used in human T-cell lymphomas, and we show that the C-terminal portion of RAG2 is required for this. This type of kinetic analysis permits us to analyze only the catalytically active RAG complex, in contrast to all other methods, which are unavoidably confounded by mixture with inactive RAG complexes. Moreover, we can distinguish the two major features of any enzymatic catalysis: the binding constant (K D ) and the catalytic turnover rate, k cat . Beyond a minimal essential threshold of heptamer quality, further suboptimal heptamer deviations primarily reduce the catalytic rate constant k cat for nicking. Suboptimal nonamers reduce not only the binding of the RAG complex to the recombination site (K D ) but also the catalytic rate constant, consistent with a tight interaction between the RAG complex and substrate during catalysis. These features explain many aspects of RAG physiology and pathophysiology.T he most fundamental aspect of any site-specific recombination process is how it achieves its site specificity, and this is also relevant for all nuclear sequence-specific proteins, including transcription factors. V(D)J recombination is the only truly sitespecific recombination process in multicellular eukaryotes, because Ig class switch recombination is a regionally specific process (22). V(D)J recombination relies on RAG1 and RAG2 forming a complex that recognizes recombination signal sequences (RSSs) composed of a palindromic heptamer (5=-CACAGTG) and an AT-rich nonamer (5=-ACAAAAACA) (13,33,34). The heptamer and nonamer are separated by either a 12-or a 23-bp spacer, thereby specifying the 12RSS or the 23RSS. A single recombination event is initiated between one 12RSS and one 23RSS when double-strand breaks (DSBs) are created directly 5= of the heptamer. The first step in creating these DSBs occurs when the RAG complex creates a nick at each of these RSS sites, followed by RAG use of the 3=-OH at that nick as a nucleophile to attack the strand opposite of the nick. This results in a hairpin at one DNA end (the coding end) and a blunt DNA end (5=-P and 3=-OH) at the RSS end (32,35).This DNA chemistry can be enzymatically catalyzed by segments of RAG1 and RAG2 that are called the core regions (designated with a c) (13,33,34). Enzymatic studies using both full-length (designated with an f) RAG1 and RAG2 have been rare because of the extremely poor solubility of these proteins (12, 37). The first report to examine the enzymatic properties of both fRAG1 and fRAG2 showed that this complex has poor cleavage activity at the RSS; however, it was not c...

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Section: Resultsmentioning

confidence: 99%

Mechanistic Basis for RAG Discrimination between Recombination Sites and the Off-Target Sites of Human Lymphomas

Shimazaki

Askary

Swanson

et al. 2012

Molecular and Cellular Biology

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“…However, there is some indirect evidence of a similar mechanism in mammalian somatic cells when transduced with lentiviral vectors. In fact, reduced expression of the introduced transgene was observed during differentiation in a murine model and silencing was found to be the result of DNA methylation of the promoter of the lentivirus driven August 12, 2015|Volume 4|Issue 3| WJV|www.wjgnet.com 229 [229] , SET7 (mono) [230] , MLL [231] , SMYD2 [232] LSD1 (mono and di) [233] , JARID1A/ KDM5A JARID1B/ KDM5B (di and tri) [234] CHD1 [235] , RAG2 [236] , TAF3 [237] , BPTF [238] , BHC80 [239] , ING FAMILY [240] , PYGO2 [241] [ [244] SIRT6 [245] BRD4 [246] [247]…”

Section: H3k27me3 or H3k9me3mentioning

confidence: 99%

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

Méndez

2015

WJV

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“…Since their discovery, the full-length RAG1 and RAG2 proteins have proven difficult to isolate and study in vitro (1). However, core domains (referred to as RAG1c and RAG2c) have been identified by removing a large region from the N terminus of RAG1 (which includes an E3 ubiquitin ligase domain) and a large region from the C terminus of RAG2 (which includes a plant homeodomain) (3,4). These core proteins have been shown to tetramerize to form RAG1/2c, which retains RSS binding, nicking, and hairpin formation activities (5).…”

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confidence: 99%

Single-molecule analysis of RAG-mediated V(D)J DNA cleavage

Lovely

Brewster

Schatz

et al. 2015

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

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The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. We have developed singlemolecule assays to examine RSS binding by RAG1/2 and their cofactor high-mobility group-box protein 1 (HMGB1) as they proceed through the steps of this reaction. These assays allowed us to observe in real time the individual molecular events of RAG-mediated cleavage. As a result, we are able to measure the binding statistics (dwell times) and binding energies of the initial RAG binding events and characterize synapse formation at the single-molecule level, yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage on forming the synapse. Interestingly, we find that the synaptic complex has a mean lifetime of roughly 400 s and that its formation is readily reversible, with only ∼40% of observed synapses resulting in cleavage at consensus RSS binding sites. The recombination-activating genes, RAG1 and RAG2, encode proteins that carry out V(D)J recombination by bringing a 12RSS and a 23RSS together into a paired (or synaptic) complex, nicking the RSS sites adjacent to the heptamer, and converting each nick into a double-strand break, leaving a hairpin at the coding end (Fig. 1). The hairpin is created by nucleophilic attack on the opposing strand by the 3′ hydroxyl group at the nick in a transesterification reaction. After RAG1/2 forms hairpins, it recruits the nonhomologous end joining machinery to repair the ends (1, 2). Since their discovery, the full-length RAG1 and RAG2 proteins have proven difficult to isolate and study in vitro (1). However, core domains (referred to as RAG1c and RAG2c) have been identified by removing a large region from the N terminus of RAG1 (which includes an E3 ubiquitin ligase domain) and a large region from the C terminus of RAG2 (which includes a plant homeodomain) (3, 4). These core proteins have been shown to tetramerize to form RAG1/2c, which retains RSS binding, nicking, and hairpin formation activities (5). High-mobility group-box protein 1 (HMGB1) acts as a cofactor and increases RAG1/2c affinity for the 23RSS (6). HMGB1 is required for paired complex formation and efficient conversion of RAG-mediated nicks into hairpins (7-10).Current in vitro assays that capture the paired complex with RAG1/2c generally place a 12RSS and a 23RSS on two different DNA molecules, typically short oligonucleotides. However, in vivo, antigen receptor loci are assembled using RSSs on the same DNA molecule (1, 11). One prior study captured RAG1/2c and HMGB1 bound to DNA with a 12RSS and a 23RSS on the same substrate using standard bulk assays (12). However, many key mechanistic questions remain unresolved concerning how a diverse immune repertoire is generated by the RAG recombinas...

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The roles of the RAG1 and RAG2 “non-core” regions in V(D)J recombination and lymphocyte development

Cited by 52 publications

References 85 publications

Mechanistic Basis for RAG Discrimination between Recombination Sites and the Off-Target Sites of Human Lymphomas

Mechanistic Basis for RAG Discrimination between Recombination Sites and the Off-Target Sites of Human Lymphomas

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

Single-molecule analysis of RAG-mediated V(D)J DNA cleavage

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