title: RAG transposase can capture and commit to target DNA before or after donor cleavage.
abstract: The discovery that the V(D)J recombinase functions as a transposase in vitro suggests that transposition by this system might be a potent source of genomic instability. To gain insight into the mechanisms that regulate transposition, we investigated a phenomenon termed target commitment that reflects a functional association between the RAG transposase and the target DNA. We found that the V(D)J recombinase is quite promiscuous, forming productive complexes with target DNA both before and after donor cleavage, and our data indicate that the rate-limiting step for transposition occurs after target capture. Formation of stable target capture complexes depends upon the presence of active-site metal binding residues (the DDE motif), suggesting that active-site amino acids in RAG-1 are critical for target capture. The ability of the RAG transposase to commit to target prior to cleavage may result in a preference for transposition into nearby targets, such as immunoglobulin and T-cell receptor loci. This could bias transposition toward relatively "safe" regions of the genome. A preference for localized transposition may also have influenced the evolution of the antigen receptor loci.
authors.affiliation: Department of Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: M B
authors.initials: MB
authors.lastname: Neiditch
authors.forename: G S
authors.initials: GS
authors.lastname: Lee
authors.forename: M A
authors.initials: MA
authors.lastname: Landree
authors.forename: D B
authors.initials: DB
authors.lastname: Roth
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016654:Genes, RAG-1; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D045567:VDJ Recombinases
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases; D008274:Magnesium; D002118:Calcium