Nucleic Acids Research Advance Access originally published online on February 20, 2009
Nucleic Acids Research 2009 37(7):2211-2226; doi:10.1093/nar/gkp047
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2009, Vol. 37, No. 7 2211-2226
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Full-length RAG1 promotes contact with coding and intersignal sequences in RAG protein complexes bound to recombination signals paired in cis
Department of Medical Microbiology and Immunology, Creighton University Medical Center, Omaha, Nebraska, 68178, USA
*To whom correspondence should be addressed. Tel: +1 402 280 2716; Fax: +1 402 280 1875; Email: pswanson{at}creighton.edu
Received November 13, 2008. Revised January 10, 2009. Accepted January 17, 2009.
The RAG proteins initiate V(D)J recombination by mediating synapsis and cleavage of two different antigen receptor gene segments through interactions with their flanking recombination signal sequences (RSS). The protein–DNA complexes that support this process have mainly been studied using RAG–RSS complexes assembled using oligonucleotide substrates containing a single RSS that are paired in trans to promote synapsis. How closely these complexes model those formed on longer, more physiologically relevant substrates containing RSSs on the same DNA molecule (in cis) remains unclear. To address this issue, we characterized discrete core and full-length RAG protein complexes bound to RSSs paired in cis. We find these complexes support cleavage activity regulated by V(D)J recombination's ‘12/23 rule’ and exhibit plasticity in RSS usage dependent on partner RSS composition. DNA footprinting studies suggest that the RAG proteins in these complexes mediate more extensive contact with sequences flanking the RSS than previously observed, some of which are enhanced by full-length RAG1, and associated with synapsis and efficient RSS cleavage. Finally, we demonstrate that the RAG1 C-terminus facilitates hairpin formation on long DNA substrates, and full-length RAG1 promotes hairpin retention in the postcleavage RAG complex. These results provide new insights into the mechanism of physiological V(D)J recombination.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. S. Shlyakhtenko, J. Gilmore, A. N. Kriatchko, S. Kumar, P. C. Swanson, and Y. L. Lyubchenko Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation J. Biol. Chem., July 31, 2009; 284(31): 20956 - 20965. [Abstract] [Full Text] [PDF]
|
|