Initiation of V(D)J recombination in a cell-free system

Dik C. van Gent; J. Fraser McBlane; Dale A. Ramsden; Moshe J. Sadofsky; Joanne E. Hesse; Martin Gellert

doi:10.1016/0092-8674(95)90012-8

Initiation of V(D)J recombination in a cell-free system

Dik C. van Gent, J. Fraser McBlane, Dale A. Ramsden, Moshe J. Sadofsky, Joanne E. Hesse, Martin Gellert

Research output: Contribution to journal › Article › peer-review

268 Scopus citations

Abstract

Cells performing V(D)J recombination make specific cuts in DNA at recombination signal sequences. Here, we show that nuclear extracts of pre-B cell lines carry out this specific cleavage. The products of cleavage are the same as found previously in thymocytes: fulllength, blunt, 5′-phosphorylated signal ends, and covalently sealed (hairpin) coding ends. A complete signal sequence is required. Recombinant RAG1 protein greatly increases activity and complements an inactive extract from a RAG1 (-/-) pre-B cell line. When the extracts are fractionated, cleavage activity correlates with the presence of RAG2 protein. These results suggest that RAG1 and RAG2 are components of the V(D)J recombinase.

Original language	English (US)
Pages (from-to)	925-934
Number of pages	10
Journal	Cell
Volume	81
Issue number	6
DOIs	https://doi.org/10.1016/0092-8674(95)90012-8
State	Published - Jun 16 1995
Externally published	Yes

ASJC Scopus subject areas

Biochemistry, Genetics and Molecular Biology(all)

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10.1016/0092-8674(95)90012-8

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@article{300d4fdbc38949358e70071a2ef032d9,

title = "Initiation of V(D)J recombination in a cell-free system",

abstract = "Cells performing V(D)J recombination make specific cuts in DNA at recombination signal sequences. Here, we show that nuclear extracts of pre-B cell lines carry out this specific cleavage. The products of cleavage are the same as found previously in thymocytes: fulllength, blunt, 5′-phosphorylated signal ends, and covalently sealed (hairpin) coding ends. A complete signal sequence is required. Recombinant RAG1 protein greatly increases activity and complements an inactive extract from a RAG1 (-/-) pre-B cell line. When the extracts are fractionated, cleavage activity correlates with the presence of RAG2 protein. These results suggest that RAG1 and RAG2 are components of the V(D)J recombinase.",

author = "{van Gent}, {Dik C.} and {Fraser McBlane}, J. and Ramsden, {Dale A.} and Sadofsky, {Moshe J.} and Hesse, {Joanne E.} and Martin Gellert",

note = "Funding Information: We are grateful to Naomi Rosenberg, Christopher Paige, and Gillian Wu for the generous gift of cell lines, and to Rose Mage for help in generating the RAG2 antiserum. We are greatly indebted to Thomas McCarthy, Susanna Lewis, and David Brown for their earlier work toward developing a cell-free V(D)J reaction. We thank Robert Craigie for his careful reading of the manuscript. We also wish to acknowledge continuing valuable discussions with our colleagues in the Laboratory of Molecular Biology. D. C. v. G. is supported by the European Molecular Biology Organisation, and D. A. R. by the Medical Research Council of Canada.",

year = "1995",

month = jun,

day = "16",

doi = "10.1016/0092-8674(95)90012-8",

language = "English (US)",

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T1 - Initiation of V(D)J recombination in a cell-free system

AU - van Gent, Dik C.

AU - Fraser McBlane, J.

AU - Ramsden, Dale A.

AU - Sadofsky, Moshe J.

AU - Hesse, Joanne E.

AU - Gellert, Martin

N1 - Funding Information: We are grateful to Naomi Rosenberg, Christopher Paige, and Gillian Wu for the generous gift of cell lines, and to Rose Mage for help in generating the RAG2 antiserum. We are greatly indebted to Thomas McCarthy, Susanna Lewis, and David Brown for their earlier work toward developing a cell-free V(D)J reaction. We thank Robert Craigie for his careful reading of the manuscript. We also wish to acknowledge continuing valuable discussions with our colleagues in the Laboratory of Molecular Biology. D. C. v. G. is supported by the European Molecular Biology Organisation, and D. A. R. by the Medical Research Council of Canada.

PY - 1995/6/16

Y1 - 1995/6/16

N2 - Cells performing V(D)J recombination make specific cuts in DNA at recombination signal sequences. Here, we show that nuclear extracts of pre-B cell lines carry out this specific cleavage. The products of cleavage are the same as found previously in thymocytes: fulllength, blunt, 5′-phosphorylated signal ends, and covalently sealed (hairpin) coding ends. A complete signal sequence is required. Recombinant RAG1 protein greatly increases activity and complements an inactive extract from a RAG1 (-/-) pre-B cell line. When the extracts are fractionated, cleavage activity correlates with the presence of RAG2 protein. These results suggest that RAG1 and RAG2 are components of the V(D)J recombinase.

AB - Cells performing V(D)J recombination make specific cuts in DNA at recombination signal sequences. Here, we show that nuclear extracts of pre-B cell lines carry out this specific cleavage. The products of cleavage are the same as found previously in thymocytes: fulllength, blunt, 5′-phosphorylated signal ends, and covalently sealed (hairpin) coding ends. A complete signal sequence is required. Recombinant RAG1 protein greatly increases activity and complements an inactive extract from a RAG1 (-/-) pre-B cell line. When the extracts are fractionated, cleavage activity correlates with the presence of RAG2 protein. These results suggest that RAG1 and RAG2 are components of the V(D)J recombinase.

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Initiation of V(D)J recombination in a cell-free system

Abstract

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