Differential requirements for cis and trans V(D)J cleavage: effects of substrate length.
The assembly of productive synaptic complexes is a critical, but poorly understood, regulatory step in V(D)J recombination. Several lines of evidence suggest that there may be important differences between recombination involving sites situated in cis (on the same DNA molecule) and in trans (on separate molecules). Because biochemical experiments using both purified RAG proteins and crude extracts have failed to detect trans cleavage of plasmid substrates it has been thought that there is a substantial bias against trans synapsis. In conflict with these results are more recent studies showing that purified RAG proteins can catalyze trans cleavage of short oligonucleotide substrates. Furthermore, recent experiments have detected efficient trans cleavage of plasmid substrates in vivo. We sought to investigate why these different systems yield such divergent results. We found that, unexpectedly, the ability of both purified RAG proteins and crude extracts to cleave DNA substrates in trans is a function of substrate length. Our data raise two critical issues: first, oligonucleotides, which are the most commonly used substrates to study V(D)J recombination in vitro, do not mimic the behavior of plasmid substrates; second, in the trans cleavage reaction current purified RAG systems do not accurately reflect the in vivo situation. We propose a unifying model to explain the effects of substrate length and coniguration (cis or trans) on the efficiency of synapsis.
Huye
L E
LE
Department of Immunology and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA.
Roth
D B
DB
D000818:Animals; D016466:CHO Cells; D002457:Cell Extracts; D006224:Cricetinae; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D015321:Gene Rearrangement / Q000235:genetics*; D018398:Homeodomain Proteins / Q000302:isolation & purification / Q000378:metabolism; D007133:Immunoglobulin Joining Region / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics*; D013379:Substrate Specificity
D002457:Cell Extracts; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D009838:Oligodeoxyribonucleotides; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA