Nucleic Acids Research, Vol 25, Issue 7 1375-1382, Copyright © 1997 by Oxford University Press
U Grawunder and MR Lieber
The recombination activating gene (RAG) 1 and 2 proteins are required for
initiation of V(D)J recombination in vivo and have been shown to be
sufficient to introduce DNA double-strand breaks at recombination signal
sequences (RSSs) in a cell-free assay in vitro. RSSs consist of a highly
conserved palindromic heptamer that is separated from a slightly less
conserved A/T-rich nonamer by either a 12 or 23 bp spacer of random
sequence. Despite the high sequence specificity of RAG- mediated cleavage
at RSSs, direct binding of the RAG proteins to these sequences has been
difficult to demonstrate by standard methods. Even when this can be
demonstrated, questions about the order of events for an individual RAG-RSS
complex will require methods that monitor aspects of the complex during
transitions from one step of the reaction to the next. Here we have used
template-independent DNA polymerase terminal deoxynucleotidyl transferase
(TdT) in order to assess occupancy of the reaction intermediates by the RAG
complex during the reaction. In addition, this approach allows analysis of
the accessibility of end products of a RAG-catalyzed cleavage reaction for
N nucleotide addition. The results indicate that RAG proteins form a
long-lived complex with the RSS once the initial nick is generated, because
the 3'- OH group at the nick remains obstructed for TdT-catalyzed N
nucleotide addition. In contrast, the 3'-OH group generated at the signal
end after completion of the cleavage reaction can be efficiently tailed by
TdT, suggesting that the RAG proteins disassemble from the signal end after
DNA double-strand cleavage has been completed. Therefore, a single RAG
complex maintains occupancy from the first step (nick formation) to the
second step (cleavage). In addition, the results suggest that N region
diversity at V(D)J junctions within rearranged immunoglobulin and T cell
receptor gene loci can only be introduced after the generation of
RAG-catalyzed DNA double-strand breaks, i.e. during the DNA end joining
phase of the V(D)J recombination reaction.
ARTICLES
A complex of RAG-1 and RAG-2 proteins persists on DNA after single- strand cleavage at V(D)J recombination signal sequences
Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.
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