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Conclusions : These results suggest that the 12/23 rule is enforced at or prior to cleavage and support a synapsis model for V(D)J recombination. Detection of rare cleavage events in substrates containing a single RSS or a pair of RSS with the same spacer length.

This approach provides a powerful new tool for analysis of the cleavage and joining steps of V(D)J recombination in vivo. In this study, substrates containing either a consensus 12/23 RSS pair or various deviations from the consensus were used to investigate the requirements for cleavage.

In vivo, efficient recombination requires one RSS of each spacer length, although it has been unclear whether this 12/23 rule regulates cleavage, joining, or both. Results : We describe a novel system that permits semiquantitative detection of DSB at RSS derived from V(D)J recombination substrates.

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The results show that both a 12spacer and a 23spacer RSS are required for efficient cleavage. Truncated RAG1 and RAG2 proteins, while capable of cleaving at isolated RSS in cellfree systems, also require a 12/23 RSS pair for efficient cleavage in vivo.

Background : V(D)J recombination is initiated by the introduction of doublestranded breaks (DSB) adjacent to recombination signal sequences (RSS). Each RSS contains a conserved heptamer and a conserved nonamer element separated by a 12 or 23 nucleotide spacer.

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