Evidence for Ku70/Ku80 association with full-length RAG1

Raval, Prafulla; Kriatchko, Aleksei N.; Kumar, Sushil; Swanson, Patrick C.

doi:10.1093/nar/gkn049

Cited by 48 publications

(53 citation statements)

References 54 publications

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“…S1A). MBP cRAGs (RAG1, amino acids 384 -1040; RAG2, amino acids 1-383) and full-length RAGs (FLRAG1, 1-1040 amino acids; FLRAG2, 1-527 amino acids) were purified using a mild method as described (40). Briefly, 14 plates of 293T cells were transfected with 10 g of plasmids each for MBP cRAG1/cRAG2 or cRAG1/FLRAG2 by the calcium phosphate method.…”

Section: Methodsmentioning

confidence: 99%

Cytosines, but Not Purines, Determine Recombination Activating Gene (RAG)-induced Breaks on Heteroduplex DNA Structures

Naik

Lieber

Raghavan

2010

Journal of Biological Chemistry

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The sequence specificity of the recombination activating gene (RAG) complex during V(D)J recombination has been well studied. RAGs can also act as structure-specific nuclease; however, little is known about the mechanism of its action. Here, we show that in addition to DNA structure, sequence dictates the pattern and efficiency of RAG cleavage on altered DNA structures. Cytosine nucleotides are preferentially nicked by RAGs when present at single-stranded regions of heteroduplex DNA. Although unpaired thymine nucleotides are also nicked, the efficiency is many fold weaker. Induction of single-or doublestrand breaks by RAGs depends on the position of cytosines and whether it is present on one or both of the strands. Interestingly, RAGs are unable to induce breaks when adenine or guanine nucleotides are present at single-strand regions. The nucleotide present immediately next to the bubble sequence could also affect RAG cleavage. Hence, we propose "C (d) C (S) C (S) " (d, double-stranded; s, single-stranded) as a consensus sequence for RAG-induced breaks at single-/double-strand DNA transitions. Such a consensus sequence motif is useful for explaining RAG cleavage on other types of DNA structures described in the literature. Therefore, the mechanism of RAG cleavage described here could explain facets of chromosomal rearrangements specific to lymphoid tissues leading to genomic instability. The recombination activating gene (RAG)3 complex, consisting of RAG1 and RAG2, is the nuclease responsible for V(D)J recombination, a physiological process by which immunoglobulin and T-cell receptor diversity is generated. RAGs are normally expressed in B-cells and T-cells (1). During V(D)J recombination, the variable (V), diversity (D), and joining (J) subexons are rearranged. Specific sequences present at the ends of the subexon, called recombination signal sequences (RSS) are recognized by RAGs. Each RSS consists of a conserved heptamer and nonamer, separated by a nonconserved spacer, the length of which designates RSS as a 12-signal or 23-signal. Normally during V(D)J recombination, a 12-signal pairs with a 23-signal with the help of proteins like HMGB1 (high mobility group box 1). The nick induced by RAGs during V(D)J recombination is consistently 5Ј of the heptamers (2-14). The nicked strand is then converted to a hairpin in each V, D, and J coding end by a transesterification reaction, leaving each of the signal ends blunt (15). The hairpins are then opened by the ArtemisDNAPKcs complex (16). After cleavage, the RAG complex remains tightly bound to the two signal ends and less tightly bound to the coding end, in a postcleavage complex (17,18). Finally, the complete exon coding for antibody or TCR (T-cell receptor) is generated by joining of the broken subexons by nonhomologous DNA endjoining (NHEJ) (19,20).In the recent past, studies have shown that cryptic RSS sites present elsewhere in the genome can also act as off-target sites for RAG misrecognition, leading to chromosomal translocations in lymphoid cancers such as l...

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Section: Methodsmentioning

confidence: 99%

Cytosines, but Not Purines, Determine Recombination Activating Gene (RAG)-induced Breaks on Heteroduplex DNA Structures

Naik

Lieber

Raghavan

2010

Journal of Biological Chemistry

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“…One possibility is that NHEJ factors stabilize the coding ends in the post-cleavage complex, increasing the time available for their attack by the RSS ends. In support of this, flRAG1, but not cRAG1, appears to associate with the Ku70/80 NHEJ protein in co-immunoprecipitation assays (Raval et al 2008). …”

Section: Transposition and Other Nonstandard Dna Transactionsmentioning

confidence: 85%

“…importin alpha 5, SRP1, and RAG cohort protein 2 (Cortes et al 1994)), karyopherin alpha 2 (KPNA2, a.k.a. RAG cohort protein 1 (Cuomo et al 1994)), and possibly the NHEJ factor Ku (Raval et al 2008). KPNA2 binds to a nuclear localization signal within the core and is necessary and sufficient for nuclear import of RAG1 (Spanopoulou et al 1995).…”

Section: Kpna1 Binding and Ubiquitylationmentioning

confidence: 99%

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

Jones

Simkus

2009

Arch. Immunol. Ther. Exp.

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The enormous repertoire of the vertebrate specific immune system relies on the rearrangement of discrete gene segments into intact antigen receptor genes during the early stages of B-and T-cell development. This V(D)J recombination is initiated by a lymphoid-specific recombinase comprising the RAG1 and RAG2 proteins, which introduces double-strand breaks in the DNA adjacent to the coding segments. Much of the biochemical research into V(D)J recombination has focused on truncated or "core" fragments of RAG1 and RAG2, which lack approximately one third of the amino acids from each. However, genetic analyses of SCID and Omenn syndrome patients indicate that residues outside the cores are essential to normal immune development. This is in agreement with the striking degree of conservation across all vertebrate classes in certain non-core domains. Work from multiple laboratories has shed light on activities resident within these domains, including ubiquitin ligase activity and KPNA1 binding by the RING finger domain of RAG1 and the recognition of specific chromatin modifications as well as phosphoinositide binding by the PHD module of RAG2. In addition, elements outside of the cores are necessary for regulated protein expression and turnover. Here the current state of knowledge is reviewed regarding the non-core regions of RAG1 and RAG2 and how these findings contribute to our broader understanding of recombination.

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“…A more recent report provides evidence for an interaction between RAG1 and Ku70/Ku80 (30). This interaction is also mediated by the N-terminal non-core region of RAG1 and affects signal joint formation.…”

Section: Discussionmentioning

confidence: 95%

“…Evidence for an interaction between RAG1 and Ku70/Ku80 represents the only known link between RAG and a repair factor (30). An early report showed that ␥-H2AX and NBS1 form RAG-dependent foci that correspond to actively rearranging loci in thymocytes (31).…”

mentioning

confidence: 99%

A Dual Interaction between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase

Coster

Gold

Chen

et al. 2012

Journal of Biological Chemistry

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Evidence for Ku70/Ku80 association with full-length RAG1

Cited by 48 publications

References 54 publications

Cytosines, but Not Purines, Determine Recombination Activating Gene (RAG)-induced Breaks on Heteroduplex DNA Structures

Cytosines, but Not Purines, Determine Recombination Activating Gene (RAG)-induced Breaks on Heteroduplex DNA Structures

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

A Dual Interaction between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase

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