Hairpin Coding End Opening Is Mediated by RAG1 and RAG2 Proteins

Besmer, Eva; Mansilla-Soto, Jorge; Cassard, Sylvanie; Sawchuk, Dennis J.; Brown, Greg; Sadofsky, Moshe J.; Lewis, Susanna M.; Nussenzweig, Michel C.; Cortés, Patricia

doi:10.1016/s1097-2765(00)80296-8

Cited by 121 publications

(92 citation statements)

References 85 publications

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“…Earlier studies reported that the RAGs are able to nick within a hairpin structure in Mn 2þ buffer (70) or extremely weakly in Mg 2þ buffer. (71) The RAG cleavage on non-B DNA structures occurs under physiologic (in the presence of Mg 2þ ) buffer conditions.…”

Section: Mechanism Of Rag Cleavage At Different Target Sitesmentioning

confidence: 99%

DNA structures at chromosomal translocation sites

Raghavan

Lieber

2006

BioEssays

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SummaryIt has been unclear why certain defined DNA regions are consistently sites of chromosomal translocations. Some of these are simply sequences of recognition by endogenous recombination enzymes, but most are not. Recent progress indicates that some of the most common fragile sites in human neoplasm assume non-B DNA structures, namely deviations from the Watson-Crick helix. Because of the single strandedness within these non-B structures, they are vulnerable to structure-specific nucleases. Here we summarize these findings and integrate them with other recent data for non-B structures at sites of consistent constitutional chromosomal translocations.

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Section: Mechanism Of Rag Cleavage At Different Target Sitesmentioning

confidence: 99%

DNA structures at chromosomal translocation sites

Raghavan

Lieber

2006

BioEssays

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“…It has been shown that RAG1/2 proteins can nick artificial hairpin ends in a cell-free system (29,30). To determine whether these proteins are responsible for processing hairpin ends in vivo, we analyzed RAG2 expression in various cell samples.…”

Section: Expression Of Rag2 and Mre11mentioning

confidence: 99%

“…The purified RAG1/2 proteins were found to mediate the reverse reaction of the cleavage to generate hybrid joints and open-shut products (31). Recently, two laboratories have shown that RAG1/2 proteins can nick hairpin ends (29,30). Interestingly, however, the resulting opened ends contained 5Ј overhangs, which differ from the opened coding ends made in vivo (Table I; 9).…”

Section: Figurementioning

confidence: 99%

“…RAG1 and RAG2 proteins have been shown to possess end-processing activities, including binding of recombination intermediates (27,28), nicking synthetic hairpin ends (29,30), and rejoining cleaved signal ends to coding ends (31). Recently, it has been demonstrated that the Mre11 protein, when complexed with Rad50 and the Nijmegen breakage syndrome gene product, Nbs1, exhibits several nuclease activities in vitro, such as hairpin nicking and processing of opened ends (32).…”

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confidence: 99%

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Metabolism of Recombination Coding Ends in scid Cells

Brown

Chang

2000

The Journal of Immunology

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V(D)J recombination cleavage generates two types of dsDNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although signal ends can be directly ligated to form signal joints, hairpin coding ends need to be opened and subsequently processed before being joined. However, the underlying mechanism of coding end resolution remains undefined. The current study attempts to delineate this process by analyzing various structures of coding ends made in situ from recombination-inducible pre-B cell lines of both normal and scid mice. These cell lines were derived by transformation of B cell precursors with the temperature-sensitive Abelson murine leukemia virus. Our kinetic analysis revealed that under conditions permissive to scid transformants, hairpin coding ends could be nicked to generate 3′ overhangs and then processed into blunt ends. The final joining of these blunt ends followed the same kinetics as signal joint formation. The course of this process is in sharp contrast to coding end resolution in scid heterozygous transformants that express the catalytic subunit of DNA-dependent protein kinase, in which hairpin end opening, processing, and joining proceeded very rapidly and appeared to be closely linked. Furthermore, we demonstrated that the opening of hairpin ends in scid cells could be manipulated by different culture conditions, which ultimately influenced not only the level and integrity of the newly formed coding joints, but also the extent of microhomology at the coding junctions. These results are discussed in the context of scid leaky recombination.

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“…DNA hairpin opening activities have been observed in vitro for the Mre11/Rad50/Nbs1 complex (29), which functions in the cellular response to DNA damage (30,31) for the RAG1/RAG2 proteins (32,33) and most recently, for the Artemis protein (34). Because the SCID DNA-PK deficiency is not known to affect the expression of any of these proteins, it was of interest to re-examine whether a portion of coding ends are open in SCID mice.…”

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confidence: 99%

Variable Diversity Joining Recombination: Nonhairpin Coding Ends in Thymocytes of SCID and Wild-Type Mice

Nakajima

Bosma

2002

The Journal of Immunology

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Initiation of V(D)J recombination results in broken DNA molecules with blunt recombination signal ends and covalently sealed (hairpin) coding ends. In SCID mice, coding joint formation is severely impaired and hairpin coding ends accumulate as a result of a deficiency in the catalytic subunit of DNA-dependent protein kinase, an enzyme involved in the repair of DNA double-strand breaks. In this study, we report that not all SCID coding ends are hairpinned. We have detected open Jδ1 and Dδ2 coding ends at the TCRδ locus in SCID thymocytes. Approximately 25% of 5′Dδ2 coding ends were found to be open. Large deletions and abnormally long P nucleotide additions typical of SCID Dδ2-Jδ1 coding joints were not observed. Most Jδ1 and Dδ2 coding ends exhibited 3′ overhangs, but at least 20% had unique 5′ overhangs not previously detected in vivo. We suggest that the SCID DNA-dependent protein kinase deficiency not only reduces the efficiency of hairpin opening, but also may affect the specificity of hairpin nicking, as well as the efficiency of joining open coding ends.

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Hairpin Coding End Opening Is Mediated by RAG1 and RAG2 Proteins

Cited by 121 publications

References 85 publications

DNA structures at chromosomal translocation sites

DNA structures at chromosomal translocation sites

Metabolism of Recombination Coding Ends in scid Cells

Variable Diversity Joining Recombination: Nonhairpin Coding Ends in Thymocytes of SCID and Wild-Type Mice

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