INITIATION OF V(D)J RECOMBINATION IN A CELL-FREE SYSTEM

被引:256
作者
VANGENT, DC
MCBLANE, JF
RAMSDEN, DA
SADOFSKY, MJ
HESSE, JE
GELLERT, M
机构
[1] Laboratory of Molecular Biology National Institute of Diabetes and Digestive, Kidney Diseases National Institutes of Health Bethesda
基金
英国医学研究理事会;
关键词
D O I
10.1016/0092-8674(95)90012-8
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Cells performing V(D)J recombination make specific cuts in DNA at recombination signal sequences. Here, we show that nuclear extracts of pre-a cell lines carry out this specific cleavage. The products of cleavage are the same as found previously in thymocytes: full-length, 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-a 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.
引用
收藏
页码:925 / 934
页数:10
相关论文
共 37 条
[11]  
Lewis, The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses, Adv. Immunol., 56, pp. 27-150, (1994)
[12]  
Lewis, Hesse, Cutting and closing without recombination in V(D)J joining, EMBO J., 10, pp. 3631-3639, (1991)
[13]  
McKearn, Rosenberg, Mapping cell surface antigens on mouse pre-B cell lines, Eur. J. Immunol., 15, pp. 295-298, (1985)
[14]  
Mombaerts, Iacomini, Johnson, Herrup, Tonegawa, Papaioannou, RAG-1-deficient mice have no mature B and T lymphocytes, Cell, 68, pp. 869-877, (1992)
[15]  
Oettinger, Schatz, Gorka, Baltimore, RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination, Science, 248, pp. 1517-1523, (1990)
[16]  
O'Reilly, Miller, Luckow, Baculovirus Expression Vectors: A Laboratory Manual, (1994)
[17]  
Parker, Topol, A Drosophila RNA polymerase II transcription factor contains a promoter-region-specific DNA-binding activity, Cell, 36, pp. 357-369, (1984)
[18]  
Pergola, Zdzienicka, Lieber, V(D)J recombination in mammalian cell mutants defective in DNA double-strand break repair, Mol. Cell. Biol., 13, pp. 3464-3471, (1993)
[19]  
Rathmell, Chu, Involvement of the Ku autoantigen in the cellular response to DNA double-strand breaks, Proc. Natl. Acad. Sci. USA, 91, pp. 7623-7627, (1994)
[20]  
Rosenberg, Lade, Chui, Lin, Dunn, Studier, Vectors for selective expression of cloned DNAs by T7 RNA polymerase, Gene, 56, pp. 125-135, (1987)