Initial stages of V(D)J recombination: the organization of RAG1/2 and RSS DNA in the postcleavage complex.
To obtain structural information on the early stages of V(D)J recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved recombination signal sequences (RSS). Stoichiometric and molecular mass analysis established that this signal-end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative-staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near the RAG1 N termini. These first images of the V(D)J recombinase in its postcleavage state provide a framework for modeling RAG domains and their interactions with DNA.
Grundy
Gabrielle J
GJ
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Ramón-Maiques
Santiago
S
Dimitriadis
Emilios K
EK
Kotova
Svetlana
S
Biertümpfel
Christian
C
Heymann
J Bernard
JB
Steven
Alasdair C
AC
Gellert
Martin
M
Yang
Wei
W
D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D018398:Homeodomain Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D007150:Immunohistochemistry; D058619:Maltose-Binding Proteins; D018625:Microscopy, Atomic Force; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D016624:Negative Staining; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis; D011995:Recombination, Genetic / Q000502:physiology*; D045567:VDJ Recombinases / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004247:DNA; D045567:VDJ Recombinases