Quantitative analyses of RAG-RSS interactions and conformations revealed by atomic force microscopy.
During V(D)J recombination, site specific DNA excision is dictated by the binding of RAG1/2 proteins to the conserved recombination signal sequence (RSS) within the genome. The interaction between RAG1/2 and RSS is thought to involve a large DNA distortion that is permissive for DNA cleavage. In this study, using atomic force microscopy imaging (AFM), we analyzed individual RAG-RSS complexes, in which the bending angle of RAG-associated RSS substrates could be visualized and quantified. We provided the quantitative measurement on the conformations of specific RAG-12RSS complexes. Previous data indicating the necessity of RAG2 for recombination implies a structural role in the RAG-RSS complex. Surprisingly, however, no significant difference was observed in conformational bending with AFM between RAG1-12RSS and RAG1/2-12RSS. RAG1 was found sufficient to induce DNA bending, and the addition of RAG2 did not change the bending profile. In addition, a prenicked 12RSS bound by RAG1/2 proteins displayed a conformation similar to the one observed with the intact 12RSS, implying that no greater DNA bending occurs after the nicking step in the signal complex. Taken together, the quantitative AFM results on the components of the recombinase emphasize a tightly held complex with a bend angle value near 60 degrees , which may be a prerequisite step for the site-specific nicking by the V(D)J recombinase.
Pavlicek
Jeffrey W
JW
School of Life Sciences, Center for Infectious Disease and Vaccinology, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287-5501, USA.
Lyubchenko
Yuri L
YL
Chang
Yung
Y
D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018625:Microscopy, Atomic Force; D008968:Molecular Conformation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000235:genetics*; D011995:Recombination, Genetic; D045567:VDJ Recombinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D021382:Protein Sorting Signals; C499311:RAG2 protein, human; C064658:RAG-1 protein; D004247:DNA; D045567:VDJ Recombinases